Integrity testing of hair samples

ABSTRACT

Methods for assessing the condition of keratinized structures, including hair, in particular methods to determine the condition of keratinized structures in relation to suitability for analysis of analytes of interest in a test sample, are presented. The methods comprise contacting the keratinized structure with a non-proteolytic reducing agent and an optional proteolytic agent. The methods further include inspection of the hair sample, or measurement of free protein eluted from the keratinized structure, after reduction and optional proteolysis to determine condition prior to analyte identification and quantitation by known techniques such as immunoassays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/472,252, filed Aug. 28, 2014, which claims priority to U.S.Provisional Application Ser. No. 61/871,162, filed on Aug. 28, 2013.

TECHNICAL FIELD

This disclosure relates to methods for assessing the condition ofkeratinized structures (e.g., hair), in particular assessing the degreeof damage in the hair. This disclosure also relates to methods fordetermining the presence or quantitation of analyte(s) of interest inkeratinized structures, particularly hair, in particular to methods forassessing the hair condition to determine the appropriate analyticprocess for a particular sample, or to determine whether a sample hasbeen subjected to procedures or conditions to render it invalid foraccurate analyte analysis.

BACKGROUND

The analysis of keratinized structures, particularly hair, for theidentification and quantitation of ingested analytes is a wellestablished practice (U.S. Pat. No. 5,364,642, U.S. Pat. No. 8,084,215).An important concern for hair analyte analysis is the change in haircondition induced by cosmetic treatment (e.g., bleaching, highlights,coloring or perms), heat treatment (e.g. heat of dryers, curling ironsor flat irons) or other sources such as combing, humidity, dryness,dirt, sunrays (UV and infared radiation) and pollution in theatmosphere. However, hair damage occurs mostly in the form of physicaland chemical changes as a result of bleaching, oxidative dyeing, hairrelaxing via alkaline relaxers, reducing waving and curling preparation.In addition to the cosmetic treatments named above, hair can alsoexhibit damage from mechanical actions such as excessive brushing,rubber-banding (e.g., ponytails), [12] or constant friction (e.g. bodyhair against clothing). While chemical interactions between the cosmeticagents and the analytes themselves are one aspect of such cosmetictreatments, an effect common to all is damage to the hair, especiallydisruption of the cuticular sheath resulting in increased porosity ofthe hair.

Increased porosity of the hair affects two main aspects of hairanalysis: (1) contaminants entering the hair and (2) compounds presentfrom ingestion being lost from the hair. Regarding the first aspect,contaminants such as drugs diffuse into the hair as solutes in solvents,particularly hair-swelling solvents; water, which swells normal intacthair about 14-16% within 30 minutes is the most prevalent and likelysolvent involved in real-life contamination. Porous hair can absorb10-20 times as much drug as intact hair under the same exposureconditions. The second aspect of the effects of porosity in hairanalysis, the loss of drugs from ingestion due to cosmetic damage, isvery much a function of the degree of porosity caused by the cosmeticdamage. If the porosity is extremely severe, which can result fromapplying cosmetic treatments in degrees not recommended by the cosmeticindustry, drug may be lost from the sample even before the samplereaches the laboratory. Thus, there is a need to identify such samplesas too damaged to produce a reliable result as to drug ingestion andreported accordingly.

The assessment of hair damage as a result of the aforementionedvariables is desired to provide reproducible and reliable methods fordetermining the presence of targeted analytes in hair samples. Hairdamage has been previously measured by a variety of methods includingmicrofluorometry, colorimetric staining, microscopy, scanning electronmicroscopy and Wilhelmy wettability. These methods, while effective,suffer from their time-consuming and laborious nature. For example, hairdamage can be determined by Methylene Blue staining (For Sci Int 176(2008) 23-33), but the method requires staining and slide mounting ofmany individual samples, making it incompatible with high volume hairanalyte analysis. Thus, a need exists for more rapid, high-throughputprotocols that are amenable to the screening of high quantities of hairsamples commonly found in hair analysis laboratories.

SUMMARY

This invention is based, in part, on the discovery that the amount oftarget analyte(s) retained within a keratinized structure, particularlya hair sample, may change based upon the condition of the hair sample,thus influencing the detection and quantitation of the targetanalyte(s). The invention is also based, in part, on the discovery thathair sample condition may affect the choice of hair washing methods toremove external/environmental contamination to distinguish presence ofanalyte(s) due to contamination from analyte(s) present by ingestion.

The present invention provides rapid and reliable methods fordetermining the condition of keratinized structures, particularly hair,as it relates to the analysis of analyte(s) released from thesestructures following proteolytic or non-proteolytic digestion.Accordingly, in one aspect, the invention features methods foroptimizing methods for the analysis of analytes from hair samples of anindividual (who may have been exposed (e.g., ingestion, inhalation, orinjection) to one or more analytes).

In one aspect, the disclosure provides methods for determining thepresence or absence of a drug of abuse or metabolite thereof in a hairsample of a subject comprising: providing a hair sample from thesubject; contacting the hair sample with an aqueous solution comprisinga reducing agent to result in a test sample; subjecting the aqueousportion of the test sample to denaturing conditions; measuring theabsorption of the subjected aqueous solution to determine if the sampleis suitable for further processing; and determining if an analyte ispresent or absent in a hair sample identified as suitable for furtherprocessing.

In another aspect, the disclosure provides methods for determining thepresence or absence of a drug of abuse or metabolite thereof in a hairsample comprising: providing a hair sample from a subject; determiningif the hair sample is not suitable for drug analyte testing comprising:contacting the hair sample with an aqueous solution comprising areducing agent to result in a test sample; and identifying the hair inthe test sample as not suitable for drug analyte testing when the hairin the test sample has dissolved in the test sample more rapidly thanthe hair in a similarly contacted control sample or the hair in the testsample appears softer or less rigid as compared to the hair in thesimilarly contacted control sample; and if the hair in the test sampleis not identified as not suitable for drug analyte testing, thendetermine if the drug of abuse is present or absent in the hair sample.

In yet another aspect, the disclosure provides methods for determiningthe presence or absence of a drug of abuse or metabolite thereof in ahair sample of a subject comprising: providing a hair sample;determining if the hair sample is not suitable for drug analyte testingcomprising: contacting the hair sample with an aqueous solutioncomprising a reducing agent and a protease capable of digesting keratinto result in a test sample; identifying the hair in the test sample asnot suitable for drug analyte testing when the hair in the test samplehas dissolved in the test sample more rapidly than the hair in asimilarly contacted control sample or the hair in the test sampleappears softer or less rigid as compared to the hair in the similarlycontacted control sample; and if the hair in the test sample is notidentified as not suitable for drug analyte testing, then determine ifthe drug of abuse is present or absent in the hair sample.

Also provided herein are methods for determining the presence of ananalyte in a hair sample of a subject, the methods comprising: assessingthe condition of a hair sample; and determining if the analyte ispresent in the hair sample.

The hair sample condition assessment step can be completed using analiquot of the same portion of sample that will be analyzed for analytesby methods, including, for example, such as an immunoassay, a massspectrometry technique or a chromatographic technique. The hair samplecondition assessment step can also be completed using a first portion ofthe hair sample, and a second portion of the same sample used foridentification and quantitation of the analyte(s). In one aspect, thestep of assessing the condition of the hair sample comprises providing ahair sample; contacting the hair sample with an aqueous solutioncomprising a reducing agent to result in a test sample; comparing thecondition of the hair in the test sample to a control sample; andidentifying the hair sample as damaged when the hair sample hasdissolved in the test solution more rapidly than the control sample, orthe hair sample appears soft and less rigid as compared to the controlsample, or identifying the hair sample as suitable for furtherprocessing if the condition of the hair in the test solution iscommensurate with the condition of the control sample.

In some embodiments, the methods provided herein further comprisingpurifying the test sample to separate the residual keratinized samplefrom the test solution, wherein the purification does notproteolytically cleave the keratinized structure, to result in apurified test solution, and determining if the analyte is present in thepurified test solution.

In some embodiments of the non-proteolytic methods described herein, themethods further comprise deactivating residual reducing agent present inthe test solution prior to the determining step, to result in adeactivated test solution. The agent used form the deactivating step maybe any agent suitable deactivating residual reducing agent, as long asthe deactivating agent does not proteolytically cleave the keratinizedstructure. In one embodiment, the deactivation step comprises contactingthe test solution with an aqueous solution of a metal salt, wherein themetal cation of the salt is selected from the group consisting of Cu++,Zn++, Mn++, Fe+++, Fe++, Pb++, Cd++, Hg++, Ag++, As+++, and Co++.

In another aspect, the step of assessing the condition of the hairsample comprises contacting the sample with an aqueous solution ofnon-proteolytic reducing agent to result in a test sample, comparing thetest sample with a control sample, and identifying the test sample asdamaged for analyte analysis when the hair sample has dissolved, appearssoft, or is less rigid in comparison to the control sample.

In yet another aspect, the step of assessing the condition of the hairsample comprises contacting the sample with an aqueous solution ofnon-proteolytic reducing agent to result in a test sample, measuringprotein eluted from the sample using established techniques (Lowry,Bradford assay), comparing protein eluted from the test sample with acontrol sample, and identifying the test sample as damaged for analyteanalysis when protein has eluted from the test sample.

In some embodiments, the step assessing the condition of the hair samplecomprises providing a hair sample; contacting the hair sample with anaqueous solution comprising a reducing agent to result in a test sample;denaturing the protein in the test sample; estimating the degree ofdamage to hair by absorption spectrophotometry of the test sample afterprotein denaturation to identify hair samples suitable for furtherprocessing; and selecting methods for determining an analyte is presentin a hair sample based on the condition of the hair.

In some embodiments, the step assessing the condition of the hair samplecomprises providing a hair sample; contacting the hair sample with anaqueous solution comprising a reducing agent to result in a test sample;denaturing the protein in the test sample; estimating the degree ofdamage to hair in the test sample by absorption spectrophotometry toidentify hair samples suitable for further processing; and determiningif an analyte is present in a hair sample identified as suitable forfurther processing.

Evaluating the degree of damage to the hair can be accomplished byabsorption spectrophotometry at a wavelength of 190 nm (A₁₉₀) to 380 nm(A₃₈₀) (e.g., A₁₉₀, A₂₀₀, A₂₄₀, A₂₈₀, A₃₀₀, A₃₄₀ or A₃₈₀). In oneembodiment, evaluating the degree of damage to the hair is performed byabsorption spectrophotometry at a wavelength of 380 nm (A₃₈₀). Formethods performed by absorption spectrophotometry at a wavelength of 380nm (A₃₈₀), samples having A₃₈₀ value above a threshold value (e.g., 0.35or greater) can be identified as not being suitable for furtherprocessing (e.g., not being suitable for use in determining if ananalyte is present in the hair sample). Conversely, samples having A₃₈₀value below a threshold value (e.g., 0.35 or less) can be identified asbeing suitable for further processing (e.g., suitable for use indetermining if an analyte is present in the hair sample).

In some embodiments, the denaturing step comprises adjusting the pH ofthe test sample to a pH of 4.0 or less, 3.5 or less, 3.0 or less, or 2.0or less.

In some embodiments, the denaturing step comprises adding an organicsolvent to the test sample. Exemplary organic solvents for use in thedenaturing step include, for example, an organic solvent selected fromthe group consisting of methanol, ethanol, propanol, 2-propanol,acetone, acetonitrile, and mixtures thereof. In another embodiments, thedenaturing step comprises adding trichloroacetic acid (e.g.,2,2,2-Trichloroacetic acid trichloroacetic acid, acetic acid, orsulfosalicyc acid to the test sample. In some embodiments, thedenaturing step comprises subjecting the hair sample to high heat orhigh acid. In some embodiments, the non-proteolytic reducing agent isselected from the group consisting of 2,3 dihydroxybutane-1,4-dithiol(“DTT”), 2,3 dihydroxybutane-1,4-dithiol (“DTE”), thioglycolate,cysteine, sulfites, bisulfites, sulfides, bisulfides,tris(2-carboxyethyl)phosphine (“TCEP”), or mixtures thereof. Forexample, the non-proteolytic reducing agent can be DTT or DTE. In someembodiments, the aqueous solution comprises 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5% or 3.0% of thereducing agent.

The pH at which the contacting step or the determining step can beperformed is between about 7.0 and 10.5, between about 8.8 and 10.5,between about 8.8 and 9.7, between about 8.8 and 9.5, or between about9.4 and 9.7.

In one aspect, the temperature at which the contacting step or thedetermining step can be performed is between about 20° C. and about 40°C.; and for a time period of about 0.5 hours to 12 hours. In oneembodiment, the contacting step occurs for a time period of about 1.5hours to about 2 hours.

In one aspect, modification of the test sample to denature proteincomprises adjustment of pH by addition of acid to a value ofapproximately 2. In another aspect, organic solvents such as methanol,ethanol and acetonitrile are added denature protein.

In one aspect, the step of assessing the condition of the hair samplecomprises contacting the sample with an aqueous solution ofnon-proteolytic reducing agent and a protease suitable for digestion ofkeratin, comparing the test sample to a control sample, and identifyingthe hair sample as damaged for analyte analysis when the sample hasdissolved in the test solution or the hair sample appears soft and lessrigid as compared to the control.

In another aspect, the step of assessing the condition of the hairsample comprises contacting the sample with an aqueous solution ofnon-proteolytic reducing agent and a protease suitable for digestion ofkeratin, comparing the test sample to a control sample, and identifyingthe hair sample as suitable for analyte analysis when the hair sampleappears similar to the control.

In some embodiments, the step of assessing the condition of the hairsample comprises providing a hair sample; contacting the hair samplewith an aqueous solution comprising a reducing agent and a proteasecapable of digesting keratin to result in a test sample; comparing thecondition of the hair in the test sample to a control sample; andidentifying the hair sample as damaged when the hair sample hasdissolved more rapidly in the test solution than the control sample, orthe hair sample appears soft and less rigid as compared to the controlsample, or identifying the hair sample as suitable for furtherprocessing if the rate of dissolution in the test solution iscommensurate with the rate of dissolution in the control sample. In someembodiments, the condition of the hair is determined based on degree ofdissolution of the hair in the test solution.

In one aspect, the condition of the test sample in proteolytic solutionis determined by one or more factors selected from the group consistingof softness, rigidity and degree of dissolution/disintegration. In oneembodiment, the condition of the sample for analyte analysis is assessedby visual inspection.

In one aspect, the protease is selected from the group consisting ofpapain, chymopapain and proteinase K.

For the proteolytic methods disclosed herein, hair sample condition isassessed by visual inspection at predetermined time intervals followingthe contacting step. In one embodiment, the predetermined time intervalsare about 10 minute, about 15 minute, about 20 minute and about 30minute intervals. The hair sample degree of damage corresponds to hairsample dissolution speed; this information can be used to determineappropriate alternate wash procedures applicable to the sample todiscern external/environmental analyte contamination from analyteingestion.

In some embodiments, the proteolytic methods disclosed herein furthercomprise identifying the hair sample as being previously treated with acellophane hair treatment if a dye is visible in the test solutionwithin about 5 minutes of contacting the hair sample with an aqueoussolution comprising a reducing agent and a protease. In otherembodiments the proteolytic methods disclosed herein, further compriseidentifying the hair sample as being previously treated with potassiumhydroxide if the hair sample fails to dissolve after a time period ofabout 120 minutes.

In yet another embodiment, the proteolytic methods disclosed hereinfurther comprise identifying the hair sample as being previously treatedwith a cellophane hair treatment if a dye is visible in the testsolution within about 5 minutes of contacting the hair sample with anaqueous solution comprising a reducing agent and a protease.

The step of determining whether an analyte is present or not present canbe performed using an enzyme immunoassay specific for the analyte (e.g.,using an antibody specific for the analyte), using a mass spectrometrytechnique, or using a chromatographic technique.

In some aspects, the determining step comprises (a) providing anoptionally washed hair sample; (b) contacting the hair sample with anaqueous solution of a reducing agent to result in a test solution; and(c) determining if the analyte is present in the test solution.

In some embodiments, the method further comprises determining the amountof analyte present, if the analyte is present. For example, the amountof the analyte present can be determined quantitatively.

The analyte to be detected can be a drug of abuse or metabolite thereof,a prescription medicine or metabolite thereof, a pain medication ormetabolite thereof, a nutrient, or an endogenous analyte, or a salt formof any of the foregoing. For example, the drug of abuse or metabolitethereof is selected from the group consisting of: cocaine,benzoylecgonine, cocaethylene, norcocaine, PCP, amphetamine,methamphetamine, cannabinoid, THC, carboxy-THC, heroin, codeine,morphine, 6-acetylmorphine (6-MAM), oxycodone,3,4-methylenedioxyamphetamine (MDA); and 3,4methylenedioxymethamphetamine (MDMA). The drug of abuse of metabolitethereof, prescription medicine or metabolite thereof, or pain medicationor metabolite thereof, including, for example, an opioid, cannabinoid,NSAID, steroid, amphetamine, benzodiazepine, barbiturate, tricyclic, orephedrine or metabolite thereof.

The analyte to be detected can be a trace metal selected from the groupconsisting of iron, magnesium, lithium, zinc, copper, chromium, nickel,cobalt, vanadium, arsenic, molybdenum, manganese and selenium.

In some embodiments, the methods disclosed herein further compriseindicating the hair sample as unsuitable for determining if the analyteis present in the test solution when the hair sample has been identifiedas damaged. For hair samples identified as damaged, the methods canfurther comprise requesting a new hair sample from the subject.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the presently described methods, suitablemethods and materials are described below. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

Other features and advantages will be apparent from the followingdetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph demonstrating hair samples following treatmentwith a reducing agent.

FIGS. 2A-2F are a series of photographs demonstrating hair samples at15, 30, 45, 60, 75 and 90 minutes following treatment using aproteolytic digestion.

FIG. 3 is a photograph demonstrating a hair sample subjected to KOHtreatment prior to treatment using a proteolytic digestion (left-handtube) and a control sample subjected treatment using a proteolyticdigestion (right-hand tube).

FIG. 4A is a photograph demonstrating a hair sample subjected tocellophane treatment prior to treatment using a proteolytic digestion(left-hand tube) and a control sample subjected to treatment using aproteolytic digestion (right-hand tube).

FIG. 4B is a photograph demonstrating a hair sample subjected to apermanent dye treatment prior to treatment using a proteolyticdigestion.

FIG. 5 is graph showing the correlation of protein concentration in thesupernatant of a test hair sample following protein denaturation of atest sample after proteolytic digestion (30 minute digestion).

FIG. 6 is a graph demonstrating the correlation of hair integrity values(scale 1-8) verses absorbance (A₃₈₀) following non-proteolytictreatment.

FIG. 7 is a graph demonstrating the correlation of hair integrity values(scale 1-8) verses absorbance (A₃₄₀) following protein denaturation of atest sample after proteolytic treatment.

DETAILED DESCRIPTION

In accordance with the present invention, methods are provided forevaluating the condition, also termed integrity, of a hair sample beingtested for analyte(s) of interest. The methods provided herein permithair sample characterization to evaluate if the hair sample is intact(of high integrity), and suitable for analyte testing, or if the samplehas been sufficiently damaged (of low integrity) to make it unsuitablefor analyte testing.

Accordingly, provided herein are methods for determining the presence ofan analyte in a keratinized structure of a subject, the methodscomprising: assessing the condition of a keratinized structure sample;and determining if the analyte is present in the keratinized structuresample. In some embodiments, the keratinized structure is hair. Thus,provided herein are methods for determining the presence of an analytein a hair sample of a subject, the methods comprising: assessing thecondition of a hair sample; and determining if the analyte is present inthe hair sample.

In some embodiments, the hair is washed repeatedly, e.g., two, three,four, five, six, seven, eight, or more times. Hair washing can beperformed by any suitable method as is known by those of skill in theart, including without limitation using a method as described in, e.g.,Baumgartner and Hill, Sample Preparation Techniques, Forensic ScienceInt. 63 (1993) 121-135. A hair sample can be washed in any appropriatebuffer, including, for example, about 0.005M to about 0.2 M phosphatebuffer (e.g., about 0.075M, 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M,0.07M, 0.08M, 0.09M, 0.1M, 0.15M, 0.175M) at a pH of from about 4.5 toabout 10.5 (e.g., about 4.5 to about 6.5, about 5.5 to about 6.5; about5.7 to about 6.2; about 5.9 to about 6.1; about 5.8 to about 7.5; about5.8 to about 8; about 6 to about 9; about 5.5 to about 9.5; about 5.8,about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about6.6, about 6.7, about 6.8, about 6.9, or about 7.0 pH). In someembodiments, the hair is washed in about 0.01M phosphate buffer about pH6.0. The hair can be washed in the suitable buffer at a temperature ofabout 25 to about 45° C., e.g., about 25 to about 32; about 28 to about32; about 30 to about 40; about 25 to about 35° C.

In these methods, a hair sample is first collected from a subject, e.g.,a subject who may have been exposed to a particular analyte or issuspected of having been so. As used herein, the term “analyte” refersto any compound, whether endogenously produced or exogenously introducedin a subject.

Thus, in some embodiments, an analyte of interest can be exogenouslyintroduced in the subject, i.e., not normally present in the subject,but introduced through an exogenous method, such as via inhalation,parenteral administration (e.g., IV, transdermal, subcutaneous, or IMroutes), or ingestion (e.g., oral, buccal, or transmucosal routes). Asused herein, a metabolite or degradation product of an exogenouslyintroduced analyte is an exogenous analyte of interest, despite the factthat it is endogenously made in vivo in a subject, because it wasderived from an exogenously introduced analyte.

As used herein, the phrases “determine the presence” and “determiningthe presence” mean determining whether or not an analyte is present.Thus, if an analyte is determined to be absent, such an activity wouldstill be encompassed by the phrases.

As used herein, a hair sample in test sample “dissolves” or “hasdissolved” “more rapidly” than another hair sample (e.g., a controlsample) when the hair sample in test sample dissolves in about 5% toabout 95%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,or about 95%, of the time it takes for the hair in the control sample todissolve.

The terms “control hair sample” or “hair in a control sample” and thelike are used interchangeably and refer to untreated hair (e.g., hairsamples with no prior cosmetic treatments) or hair samples previouslydetermined as not being excessively damages or porous.

An analyte can be any chemical, including drugs of abuse, toxicchemicals, environmental chemicals (e.g., pesticides, herbicides,insecticides), petroleum products, natural products, organic compounds,nutrients, prescription or over-the-counter medications (e.g., painmedications, steroids, narcotics, NSAIDS), or metabolites, derivatives,or breakdown products of any of the foregoing.

In some embodiments, an analyte of interest can be an exogenouslyintroduced drug-of abuse, prescription medication, pain medication,organic compound, nutrient, metal, toxic chemical, pesticide, or ametabolite or degradation product thereof. Examples of drugs of abuse,pain medications, or prescription medications, or metabolites thereof,include an opioid, cannabinoid, NSAID, steroid, amphetamine,benzodiazepine, barbiturate, tricyclic, or ephedrine, or metabolitethereof.

Specific examples include: cocaine (and metabolites benzoylecgonine,cocaethylene, and norcocaine), opioids and metabolites thereof(morphine, heroin, 6-monoacetylmorphine, diacetylmorphine, codeine,oxycodone, hydrocodone, hydromorphone, oxymorphone, and methadone),cannabinoids, phencyclidine (PCP), amphetamines, methamphetamines, MDMA(ecstasy, methylenedioxy methamphetamine), MDA(methylenedioxyamphetamine), marijuana (and THC and carboxy-THCmetabolites), propoxyphene, meperidine, benzodiazepines, carisoprodol,tramadol, fentanyl, buprenorphine, naltrexone, tricyclics, nicotine (andits metabolite cotinine), eve (methylenedioxy-ethylamphetamine),flunitrazepam, lysergic acid (LSD), digoxin, methylphenidate,acetaminophen, salicylates, fluoxetine, sertraline, dextromethorphan,ephedrine, phenethylamines, pseudoephedrine, and synephrine. Pesticidesinclude, without limitation, parathion, malathion, chlorpyrifos,diazinon, dichlorvos, and tetrachlorvinphos.

In other embodiments, an analyte of interest is endogenously produced,e.g., in an amount that correlates with the presence or absence of adisease state or metabolic state of a subject. Examples of endogenousanalytes include fatty acid esters (e.g., as markers of alcoholconsumption); chromium (e.g., as measure of glucose tolerance and type 2diabetes); glucose (e.g., as measure of glucose tolerance and type 2diabetes); and glycosyl groups (e.g., as a measure of chronichyperglycaemia).

In one aspect, a hair sample is first collected from a subject suspectedof having been exposed to, or having ingested, a particular analyte.Preferably, the hair sample is first washed by known methods to removeanalyte or other drug or chemical which may have been deposited on thesurface of the hair by external contact rather than by actualconsumption. The sample is then subjected to proteolytic ornon-proteolytic digestion methods, so as to effectuate trapped analyterelease.

In one aspect, the hair sample is subjected to non-proteolytic methodsto release entrapped analytes. For the non-proteolytic digestionmethods, treatment of the hair sample does not include contacting thehair sample with one or more proteolytic enzymes, such as papain,chymopapain, and proteinase K. Thus, the treatment method does notproteolytically cleave peptide (amide) bonds in the structure, e.g., notcleave them substantially. In some embodiments, the method reduces,e.g., reduces substantially, disulfide bonds present in the hair samplebut does not cleave peptide bonds (e.g., does not cleave themsubstantially) in the sample. Typically, the treatment method comprisesa reducing step, an optional deactivation step, and an optionalpurification (e.g., separation, filtration, or centrifugation) step.

In the non-proteolytic reducing step, the sample is contacted with asolution of a reducing agent (reducing solution), such as Dithiothreitol(“DTT”), so as to reduce inter- and intra-molecular disulfide bonds inthe keratin macrostructure, thereby releasing entrapped analyte. In someembodiments the keratinized structure sample can be contacted with anon-proteolytic reducing solution consisting essentially of the reducingagent, or can be contacted with a reducing solution that does notcomprise a proteolytic enzyme. In some embodiments, the contacting stepdoes not result in the substantial breakage of peptide backbone bonds(i.e., amide bonds) in the keratin polypeptide chains.

According to one aspect, the hair sample can range in size from about 2to about 12 mg per mL of reducing agent solution, e.g., from about 3 toabout 10 mg, from about 4 to about 8 mg, from about 7 to about 15 mg,from about 5 to about 10 mg, or from about 8 to about 14 mg per mL ofreducing agent solution. The sample can be first washed by known methodsto remove analytes or contaminants which may have been deposited on thesurface by external contact rather than by actual consumption.

In order to determine the presence and optionally the concentration ofone or more analytes, a test sample can be taken from the treated hairsample, either after the contacting step with the reducing solution orafter the optional deactivation step. The sample can be removeddirectly, after the optional deactivation step, or after an optionalpurification step (e.g., separation, centrifugation, or filtration) toremove residual reduced keratinized sample.

The reducing agent for inclusion in the non-proteolytic reducingsolution can be any reducing agent capable of reducing disulfide bondsin keratinized structures, e.g., hair. Typical examples include DTT (2,3dihydroxybutane-1,4-dithiol) or its isomer DTE (2,3dihydroxybutane-1,4-dithiol), thioglycolate, cysteine, sulfites,bisulfites, sulfides, bisulfides, 2-mercaptoethanol or TCEP(tris(2-carboxyethyl)phosphine), or salt forms of any of the foregoing.

Typically, the concentration of the non-proteolytic reducing agent inaqueous solution during the contacting step is about 1 to about 20 g/L,e.g., about 1 to about 15, about 2 to about 14, about 5 to about 15,about 10 to about 18, about 3 to about 12, about 4 to about 8, g/L. Asone having ordinary skill in the art would recognize, the amount ofreducing agent can vary based on the length of the reaction time and thedetection methodology to be used.

In some embodiments, the methods may be conducted at or near roomtemperature and near neutral pH. For example, the method may beperformed at a temperature of between about 20° C. and 60° C. (e.g.,about 20, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, or 60° C.) and at a pH between about pH 5 and about 10.5. In someembodiments, the pH of the method is between about 8.8 and 9.7 (e.g.,8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.45, 9.5, 9.55, 9.6, 9.65) and the methodoccurs at a temperature of about 37° C. In other embodiments, e.g.,where an analyte of interest or metabolite or degradation productthereof is sensitive to basic pHs, a lower pH can be used, e.g., betweenabout 5 to about 8.7 (e.g., about 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4,6.6, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, or 8.7). Appropriatereaction conditions, including reaction temperature, time, and pH, canbe readily determined by those having ordinary skill in the art.

DTT and DTE are particularly useful as reducing agents. It has beenfound that the use of DTT or DTE in the described processes results inrelease of the entrapped analytes within a relatively short period oftime (depending on the amount and type of keratinized sample), e.g., inabout 0.5 to about four hours, or about 1 to about 3 hours, or about 1.5to about 2.5 hours. In certain embodiments, treatment for about 2 hoursis sufficient, e.g., for about 5-15 mg of keratinized sample such ashair.

Once the hair sample has been treated with a solution of anon-proteolytic reducing agent for a time sufficient to releaseentrapped analytes, e.g., in about 0.5 to about four hours, or about 1to about 3 hours, or about 1.5 to about 2.5 hours, or about 2 hours, thecondition of the hair sample for analyte analysis is assessed.

In one aspect, the condition for analyte analysis after non-proteolyticdigestion is assessed visually. Hair samples that exhibit disintegrationafter non-proteolytic reduction as compared to the control sample (e.g.,healthy hair) can be identified as damaged for analyte analysis. Hairsamples that appear similar to the control sample (e.g., dissolution at105-120 minutes) can be identified as intact for analyte analysis. Hairsamples identified as extremely damaged may not be suitable for use insubsequent analyte detection methods. In some embodiments, methodsdisclosed herein include requesting an additional hair sample from anindividual when their hair sample has been identified as invalid forhair analyte analysis.

Alternatively, the hair samples identified as damaged may be labeled orflagged as damaged and still analyzed in downstream analyte testingprocedures. The labeling alerts the technician performing the downstreamanalyte testing procedures of the condition and that the results shouldbe monitored and interpreted accordingly. Subsequent washing proceduresapplied to analyte-positive samples will then be selected based on thehair condition.

In practice, hair samples may be treated with a solution of anon-proteolytic reducing agent (e.g., DTT) under conditions that allowfor digestion of the disulfide bonds, and therefore release of analytesfrom the hair sample, in about 2 hours. After 2 hours, the hair samplesare observed for extent of visible degradation of the hair for analyteanalysis. In one embodiment, hair samples that appear softened anddisintegrated at this 2-hour time-point are identified as damaged foranalyte analysis. Such samples may be reported as invalid for analysis.In another embodiment, samples identified as damaged or porous can belabeled damaged, and then used in subsequent analyte detection methods.

In more detail, hair samples that have been treated with a solution ofproteolytic reducing agent can be rated on a scale of 1 to 8, 8 beingintact hair or “strong” hair (control tubes). An estimate of the stateof the weak or porous hair appearance may be 1-2 (completelydisintegrated hair), 3-6 (at least partially disintegrated hair withsome intact hair), and 7-8 (minimal or no disintegration). Sampleshaving a rating of 1-2 can be identified as porous and damaged foranalyte analysis. These samples may be reported as invalid for analysis.Samples having a rating of 4-6 can be identified as damaged or porous,and then used in subsequent analyte detection methods. Such samples canbe labeled or flagged as damaged or porous, alerting the technicianconducting subsequent analyte detection methods.

Once the analyte(s) have been released into the solution mixture,residual active reducing agent can be optionally deactivated by methodsknown to those having ordinary skill in the art, including simplywaiting a sufficient period of time for deactivation to naturally occur.Typically this time period is from about 2 to about 14 hours afterinitial contact of the reducing agent with the hair sample, depending onthe concentration and amount of reducing agent utilized, the pH,temperature, size of sample, etc. As with the contacting step, thedeactivation step is performed in the absence of a proteolytic enzyme(e.g., in a solution consisting essentially of the deactivation agent,or in a solution that does not comprise a proteolytic enzyme).

Alternatively, as known to those having ordinary skill in the art,residual reducing agent can be deactivated with the addition of certainmetal ions, typically in the form of metal salts, to the reducingsolution. The addition of low amounts, e.g., from about 0.1 to about 1.0g/L in the final sample solution, of such metal salts to the reducingsolution after contacting it with the sample can significantlyaccelerate the time in which the reduced sample can be subjected to theanalyte detection method, since it is not necessary to wait for thereducing agent to deactivate on its own. Most effective are certainmetal salts which do not precipitate out of the solution afterchemically linking with, and deactivating the reducing agent, such asDTT or DTE. It can be useful to avoid precipitation in the reducingsolution because such precipitation could result in a loss of analyte byadsorption to the precipitate or entrapment therein, or could causeinterference by particulate obstruction of optical reading methods.

In addition to Cu++ salts (e.g., copper sulfate) as described in U.S.Pat. Nos. 5,466,579 and 5,324,642, salts of Zn++ (e.g., zinc sulfate andzinc nitrate); Mn++ (e.g., manganese sulfate); Fe+++ (e.g., ferricsulfate and ferric chloride); and Fe++ (e.g., ferrous sulfate) areeffective. Also effective are salts of Pb++ (e.g., lead acetate and leadnitrate); Cd++ (e.g., cadmium chloride); Hg++ (e.g., mercuric chloride);Ag++ (e.g., silver nitrate); and Co++ (e.g., cobalt chloride). See,e.g., U.S. Pat. Nos. 6,022,693 and 6,350,582.

In certain embodiments, a salt of arsenite, such as sodium arsenite(NaAsO2), may be utilized to remove residual reducing agent (e.g., DTTor DTE) by formation of a precipitable compound. Typically, 100microliters of a 100 mg/mL solution of sodium arsenite is added to 1 mLof hair digest solution (final concentration of about 10 g/L) toeffectuate the deactivation of the reducing agent. However, arsenite isnot preferred because a precipitate can develop, thereby potentiallyadsorbing or entrapping analyte.

Typically, from about 0.1 to about 1 mg (e.g., about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg) of a metal salt in solution can beadded to about 0.8 to about 1.6 mL (e.g., about 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5 or 1.6 mL) of reducing solution at a time period fromabout 1 to about 5 (e.g., about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5)hours after contacting the sample with the reducing solution. Typically,the deactivation is rapidly complete, e.g., in less than about 30minutes, such as in less than about 20 mins., less than about 10 mins.,less than about 5 mins., or less than about 2 mins.

Additional information on the methods described herein, includingdeactivation methods, can be found in U.S. Pat. No. 8,084,215, which ishereby incorporated by reference in its entirety.

In another aspect, the hair sample is subjected to simultaneousproteolytic and non-proteolytic (reduction) methods to release entrappedanalytes. In one embodiment, the simultaneous treatment includescontacting the hair sample with one or more proteolytic enzymes, such aspapain, chymopapain, and proteinase K, and a reducing agent, such as DTTor DTE. Thus, the treatment method comprises contacting the hair samplewith a reducing agent solution, so as to reduce inter- andintra-molecular disulfide bonds in the keratin macrostructure, therebyreleasing entrapped analyte, and an enzyme suitable for the dissolutionof the hair sample.

Typically, the treatment method comprises an enzymatic digestion incombination with reducing agent step, an optional deactivation step, andan optional purification (e.g., separation, filtration, orcentrifugation) step. In the proteolytic step, the sample is contactedwith a solution of an enzyme suitable for the dissolution of akeratinized structure, e.g., a proteolytic enzyme, and a reducing agent,such as Dithiothreitol (DTT), thereby releasing entrapped analyte. Formethods comprising contacting the hair sample with one or moreproteolytic enzymes, the enzyme is selected from the group consisting ofpeptidase, endopeptidase, and protease, and preferably is papain,chymopapain, or proteinase K.

A number of other proteases have been found to be effective in themethod according to the invention at low pH values (e.g., pH 7-9),namely, protease Type IV (bacterial, from Streptomyces caespitosus),Type VIII (from Bacillus subtilis), Type XI (proteinase K, fungal, fromTritirachium album), Type XIV (pronase, from Streptomyces griseus), TypeXVI (from Bacillus subtilis), Type XVIII (Newlase, from Rhizopusspecies), Type XIX (from Aspergillus sojae), Type XXI (from Streptomycesgriseus), Type XXIV (bacterial), Type XXVII (Nagarase), Type III(Prolase) and Type XXIII (from Aspergillus oryzae) (all available fromSigma Chemical Co., St. Louis, Mo.).

Once the protein of the hair sample (e.g., the keratinized structure)has been completely or at least substantially dissolved, therebyreleasing the analyte into the solution mixture, it has been found to benecessary to deactivate the enzyme and the enzyme/substrate activator(s)in order to subject the analyte to biological analytical probes such asantibodies, since the enzyme and enzyme/substrate activator(s), as notedabove, can interfere with the structural integrity of protein substancesinvolved in the analytical method.

According to one aspect, the hair sample can range in size from about 4to about 16 mg per mL of reducing agent solution, e.g., from about 5 toabout 12 mg, from about 6 to about 10 mg, from about 7 to about 15 mg,from about 5 to about 10 mg, or from about 8 to about 14 mg per mL ofreducing agent solution. In a one aspect, the hair sample is keptconstant at about 5 mg/ml so as to prevent variable matrix effects insubsequently utilized protein-based analytical methods.

In some embodiments, the enzymatic digestion of the hair sample may beconducted at low temperature and near neutral pH. For example, themethod may be performed at a temperature of between about 20° C. and 40°C. (e.g., about 20, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, or 60° C.) and at a pH between about pH 5 and about 10.5. Insome embodiments, the pH of the method is between about 8.8 and 10.5(e.g., 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.45, 9.5, 9.55, 9.6, 9.7, 9.8,9.9, 10.0, 10.1, 10.2, 10.3, 10.4 or 10.45) and the method occurs at atemperature of about 37° C. Appropriate reaction conditions, includingreaction temperature, time, and pH, can be readily determined by thosehaving ordinary skill in the art.

Additional information on the methods described herein, including theoptional deactivation methods can be found in U.S. Pat. No. 6,949,344,incorporated by reference. In the proteolytic methods described above,hair samples are generally completely dissolved in about 2 hours.Damaged (e.g., porous) hair samples dissolve more quickly than healthyhair samples. To monitor the rate of dissolution, hair samplesundergoing treatment using a proteolytic digestion are observed by atechnician at 15 minute intervals. The technician observes and recordsduring the process the extent of hair dissolution or disappearance.

In one embodiment, the appearance of the hair can be rated on a scoringtable. For example, a sample that does not significantly dissolve until105-120 minutes and has no dye color is scored a 8 value, for maximalintegrity. These samples are suitable for analyte analysis. Scores forsamples with shorter digestion times are as follows: more than 70%digestion at 15 min=1; @ 30 min=2; @ 45 min=3; @ 60 min=4; @ 75 min=5; @90 min=6. If a sample shows no dissolution in 120 minutes, thisindicates that the hair has been relaxed using an alkaline treatmentmethod, such as a potassium hydroxide (KOH) treatment. Samples havingmore than 70% dissolution at 15, 30, 45, 60 or 75 minutes can be labeledas damaged or porous. Such samples may be reported as invalid/unsuitablefor analysis, or if less damaged, subjected to analytical methodsappropriate for porous hair.

In one aspect, the methods disclosed herein further comprise quantifyingthe amount of dissolved hair protein for hair samples subjected to theproteolytic and/or non-proteolytic digestion methods described herein.Amongst several methods, dissolved protein is readily determined by thecolorimetric method of Lowry et al. (1951), with reference to a standardcurve of bovine serum albumin. The colored complex is a result of acomplex between the alkaline copper-phenol reagent used and tyrosine andtryptophan residues of the protein, and can be detect byspectrophotometer at 705 nm. Protein concentration is also readilydetermined by the colorimetric method of Bradford. (distributedcommercially by BIO RAD as the BIO RAD PROTEIN ASSAY®). Correlation ofprotein concentration in the supernatant of the proteolytic digestion at30 minutes digestion is shown in FIG. 5.

Such assay methods are commonly used for assaying protein in solutionand known to those having ordinary skill in the art. Suitable proteinassay methods can be found in, e.g., Bradford, “A Rapid and SensitiveMethod for the Quantitation of Microgram Quantities of Protein Utilizingthe Principle of Protein-Dye Binding”, Analytical Biochemistry Vol. 72,pp. 248-254 (1976); Han et al., “A simple improved method for proteinextraction from human head hairs”, J. Cosmet. Sci., Vol. 58, pp. 527-534(September/October 2007); Inoue et al., “Elution of 8100A3 from hairfiber: New model for hair damage emphasizing the loss of S100A3 fromcuticle”, J. Cosmet. Sci., Vol. 51, pp. 15-25 (January/February 2000);Jeong et al., “Hair Damage and Wave Efficiency according to the Degreeof Alkalinity in Permanent Wave”, Applied Microscopy, Vol. 42(3), pp.136-141 (2012); Inoue et al., “Labile proteins accumulated in damagedhair upon permanent waving and bleaching treatments”, J. Cosmet. Sci.,Vol. 53, pp. 337-344 (November/December 2002); Zor et al.,“Linearization of the Bradford Protein Assay Increases Its Sensitivity:Theoretical and Experimental Studies”, Analytical Biochemistry, Vol.236, pp. 302-308, Article No. 0171 (1996); Tate et al., “Quantificationand prevention of hair damage”, J. Cosmet. Sci., Vol. 44, pp. 347-371(November/December 1993); Sinclair et al., “The proteomic profile ofhair damage”, British Association of Dermatologists Vol. 166 (Suppl. 2),pp. 27-32 (2012); Davis et al., “Utilizing Proteomics to CharacterizeHair and Hair Damage at the Molecular Level”, P&G beauty & grooming(undated), the disclosures of each of which are incorporated byreference in their entirety.

Once the treatment of the sample is complete (e.g., non-proteolytictreatment of the hair sample or proteolytic treatment of the hairsample), the hair sample solution may be subjected to direct analysis byart recognized analyte detection methods, including receptor assays,protein-based analytical methods such as immunoassay includingradioimmunoassay (RIA) or enzyme immunoassay (EIA), and/or instrumentalmethods such as mass spectroscopy chromatographic techniques, or atomicabsorption.

Additional information on the methods described herein, includingmethods for determining the presence of an analyte in a sample, can befound in U.S. Pat. No. 8,501,494, U.S. Pat. No. 7,629,129, U.S. Pat. No.7,618,591, U.S. Pat. No. 7,083,925, U.S. Pat. No. 7,060,453, U.S. Pat.No. 6,949,344, U.S. Pat. No. 6,537,825, U.S. Pat. No. 6,022,693, U.S.Pat. No. 5,981,204, U.S. Pat. No. 5,910,419, U.S. Pat. No. 5,324,642,U.S. Pat. No. 4,956,467, EP 2518474, WO 2014/005065, WO 2005/121793, WO2005/039381, WO 2003/031935, Harrison, R. and Fu, S., J. Forensic Sci.,Vol. 2(1): 1-8 (January 2014), Koster et al., Therapeutic DrugMonitoring, Vol. 36(2): 234-243 (April 2014), Vincenti et al.,Bioanalysis, Vol. 5(15): 1919-1938 (August 2013), Lopez-Guarnido et al.,J. Applied Toxicology, Vol. 33(8): 838-844 (August 2013), Kronstrand etal., Therapeutic Drug Monitoring, Vol. 35(3): 288-295 (June 2013),Cooper et al., Forensic Sci. Int., Vol. 218(1-3): 20-24 (May 2012),Albermann et al., Anal Bioanal Chem, Vol. 400: 175-181 (2011), Musshoff,F. and Madea, B., Anal Bioanal Chem, Vol. 388: 1475-1494 (2007),Gratacos-Cubarsi et al., Journal of Chromatography B., Vol. 834: 14-25(2006), Nielen et al., J. Chrom. B., Vol. 830: 126-134 (2006), Pascal etal., Therapeutic Drug Monitoring, Vol. 26(2): 211-214 (April 2004), Chanet al., Clinical Biochemistry, Vol. 37(6): 429-138 (June 2004), Dolan etal. Drug and Alcohol Review, Vol. 23(2): 213-217 (2004), Villain et al.,Forensic Sci. Int., Vol. 145(2-3): 117-121 (October 2004), Kintz, LegalMedicine, Vol. 5(Supplement): S29-S33 (March 2003), Baptista et al.,Forensic Sci. Int., Vol. 128: 68-78 (August 2002), Lewis et al.,Forensic Sci. Int., Vol. 84(1-3): 123-128 (January 1997), Yegles et al.,Forensic Sci. Int., Vol. 84(1-3): 211-218 (January 1997), Polettini etal., Forensic Sci. Int., Vol. 84(1-3): 259-269 (January 1997), Kintz etal., J. Forensic Sci., Vol. 40(4): 619-22 (July 1995), Kintz et al., J.Chromatography B: Biomedical Sciences and Applications, Vol. 670(1):162-166 (August 1995), Ahrens et al., J. Anal. Chem., Vol. 344: 559-560(1992), and Baumgartner et al., J. Nuclear Med., Vol. 20: 748-752(1979), which is hereby incorporated by reference in its entirety.

In particular embodiments, instrumental methods (e.g., a massspectrometry technique or a chromatographic technique) may be used toconfirm positive results obtained in immunoassay methods. Because thesemethods are not protein-based, the step of deactivation of reducingagent is not necessary. The speed and gentleness of the treatment methodand the ability to quantitate efficiency through the inclusion of a“spike,” i.e., the inclusion of a known amount of deuterated analyte,makes the presently disclosed treatment method also the method of choicefor instrumental analysis methods such as gas chromatography, liquidchromatography and mass spectrometry. The method can be used to detectthe use and prior use of any analyte of interest described previously,including drugs of abuse such as cocaine, morphine/heroin and otheropioids, cannabinoids, marijuana, phencyclidine or “PCP,” methaqualone,and amphetamines. Moreover, the method can be effective in determiningprior usage of prescription drugs such as digoxin, methadone andbenzodiazepines. It is contemplated that any analyte, particularly anyorganic analyte, present in the bloodstream of an individual which istransferred to the hair during its synthesis can be extracted andanalyzed in accordance with the methods described herein.

In some aspects, a detergent can be used to aid in the release of one ormore analytes of interest from the hair sample. Certain biologicaldetergent compounds useful for solubilizing biological membranecomponents aid in the release of the analytes at a relatively low pHwhile not interfering with reduction or subsequent analyte detection.These biological detergents can aid the in the treatment of akeratinized sample at a pH in the range of about 5 to about 10.5.Suitable detergents include bile acid detergents, such as glycocholicacid, cholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholicacid, taurodeoxycholic acid and salts thereof, including sodium salts.Other detergents for use in the methods are sulfo-betaines, such as theZwittergents® and betaines, such as Empigen BB(N-dodecyl-N,N-dimethylglycine) (all available from Calbiochem Corp., LaJolla, Calif.). Other detergents include alkylglucosides, includinghexyl-beta-D-glucopyranoside, heptyl-beta-D-glucopyranoside,octyl-beta-Dglucopyranoside, nonyl-beta-D-glucopyranoside,decyl-beta-D-glucopyranoside, dodecyl-beta-D-maltoside andoctyl-beta-D-thioglucopyranoside (OSGP). Mixtures of alkylglucosides,such as the product ELUGENT® (Calbiochem), are also effective.Particularly preferred are the bile acids cholic acid and glycocholicacid, which aid in the digestion of hair at a pH in the range of about6.3 to about 8. The deoxycholates such as deoxycholic acid andglycodeoxycholic acid are effective in aiding in the digestion of hairat a pH above about 7.

The detergents can be used in the industry standard five-drug screen forthe most common drugs of abuse in the United States, i.e., marijuana,cocaine, phencyclidine, methamphetamine and opioids, measured using themethods described herein. Thus, they do not impact any of the analytesor antibodies involved in the five-drug screen, and do not result infalse negatives or positives. The particular detergents most effectivefor use in the five-drug screen are cholate, deoxycholate, cholic acid,deoxycholic acid, octyl-beta-D-glucopyranoside andoctyl-beta-D-thioglucopyranoside. The bile acid detergents,alkylglucosides, sulfobetaines and betaines are preferred when a screenis performed that includes cocaine, opioids, phencyclidine, amphetaminesand sympathomimetic amines. In a screen solely for cocaine, thepreferred detergents are cholic acid, Zwittergents®, alkylglucoides, andN-dodecyl-N,N dimethylglycine.

In practice, the biological detergent is mixed with the aqueous reducingsolution prior to contact of the solution with the keratinized sample ata temperature range of about 30 to about 40° C. Typically, about 1-2 mgof biological detergent is added to about 1 ml of reducing solution.

Additional information on the methods described herein, including theuse of biological detergents, ion exchange resins (e.g., to removeinterfering substances), and varying pH ranges for digestion, can befound in U.S. Pat. Nos. 6,022,693 and 6,350,582, incorporated herein byreference in its entirety.

In some embodiments, the methods disclosed herein further compriseindicating the hair sample as unsuitable for determining if the analyteis present in the test solution when the hair sample has been identifiedas damaged. For hair samples identified as damaged or identified asunsuitable for determining if the analyte is present, the methods canfurther comprise requesting a new sample from the subject. The newsample can be a new keratinized sample (e.g.,) new hair sample or othersample suitable for use for testing for the presence and/or amount ofone or more analytes of interest.

Any type of sample can be tested for the presence and/or amount of oneor more analytes of interest. In certain cases, a sample contains or issuspected to contain one or more analytes of interest, such as one ormore drugs of abuse or toxic chemicals. A sample can be a bodily sampleor a non-bodily sample. A bodily sample (e.g., a hair sample) can be aspecimen obtained from an individual (e.g., a human, mouse, rat, pig,horse, monkey, rabbit, cow, sheep, or goat). A bodily sample can be atissue sample, such as a tissue sample of the brain, heart, lungs,kidneys, liver, muscle, bone, stomach, intestines, or skin. A bodilysample can be obtained by biopsy or from tissue culture. A bodily samplecan include a biological fluid such as urine, blood, plasma, serum,saliva, semen, sputum, cerebral spinal fluid, mucus, sweat, milk,vitreous fluid and the like. A bodily sample can be a keratinizedstructure, such as hair, a fingernail, or a toenail. A non-bodily samplecan be, for example, a soil or water sample, a plant sample, aninorganic material sample, or a sample from a research or manufacturingprocess.

A sample can be used as is, or can be treated to result in a finalsample for detection of the one or more analytes. For example, a samplecan be liquefied, concentrated, dried, diluted, lyophilized, extracted,fractionated, subjected to chromatography, purified, acidified, reduced,degraded, subjected to enzymatic treatment, or otherwise treated in waysknown to those having ordinary skill in the art in order to release ananalyte of interest. If desired, a sample can be a combination (pool) ofsamples, e.g., from an individual or from a manufacturing process.

A sample can be in a variety of physical states, e.g., liquid, solid,emulsion, or gel. Samples can be treated with customary care to preserveanalyte integrity. Treatment can include the use of appropriate buffersand/or inhibitors, such as inhibitors of certain biological enzymes. Onehaving ordinary skill in the art will be able to determine theappropriate conditions given the analytes of interest and the nature ofthe sample.

In some embodiments, a sample is derived from a bodily sample.

In some embodiments, a sample is derived from a keratinized structure.In some embodiments, the keratinized structure is hair.

In one aspect, the disclosure provides methods for determining if ananalyte is present in a hair sample after the hair sample has beenidentified as damaged or as having increased porosity. As shown in theexamples, it is possible to excessively wash damaged or porous hairusing an aqueous wash solution, resulting a false negative result. Thefact that some drug may wash out in a damaged or porous hair sample doesnot necessarily disqualify the sample from further analyte testing.Rather, the hair sample can be washed with a non-swelling solvent (e.g.,ethanol) prior to the assessing and/or determining steps.

In some embodiments, the methods disclosed herein further comprisesubjecting a hair identified as damaged or as having increased porosityto an ethanol washing procedure as described herein, and determining ifthe analyte is present in the hair sample. In one embodiment, theethanol was procedure comprises soaking the hair sample two, three,four, five, or six times in 90% ethanol. For example, the hair sample issoaked in 90% three times for 30 minutes and then soaked in 90% ethanolfor two 60 minute washes. The last wash sample may then be analyzed todetermine the presence of an analyte.

Additionally, the degree to which an analyte if retain within, or elutesfrom a damaged, porous hair sample depends, in part, on the analyte.Specifically, some drug analytes are retained within a damaged, poroushair sample more readily than other drug analytes. For instance, asshown in the examples, methamphetamine and metabolites thereof areretained within porous hair samples more readily than cocaine. Thus,depending on the analyte of interest, it is possible, even recommendedto complete the determining step for hair samples identified as damagedor as having increased porosity.

In some embodiments, the methods disclosed herein comprise determiningif an analyte is present in a hair sample which has been identified asdamaged or as having increased porosity for analytes which are likely tobe retained with damaged, porous hair. For analytes which do not readilyelute from damaged, porous hair., the methods may be further modified toinclude a step of subjecting a hair identified as damaged or as havingincreased porosity to an ethanol washing procedure as described herein,and determining if the analyte is present in the hair sample.

The benefits to be obtained from the presently disclosed methods aremany, including a prompt, accurate, and inexpensive determination ofprior exposure to a particular analyte, after assessment of whether thesample is in suitable condition for analysis. The method can provide arecord of consumption, or non-consumption, over very long periods oftime. Moreover, hair collection is less intrusive and less physicallyrepulsive than blood or urine collection, and samples cannot be alteredor substituted, nor can detection be evaded by short term abstention or“flushing” (excessive fluid intake) prior to a scheduled testing, e.g.,pre-employment test or annual physical examination. Samples may bestored indefinitely without refrigeration. Finally, the methodsfacilitate both screening and confirmatory assays for detecting ananalyte of interest.

The following examples are meant to be illustrative and do not limit theclaims.

EXAMPLES

Three new methods (Examples 1-3) to characterize the condition of hairsamples, the methods for which are presented below, include (1)observation of dissolution of hair in a non-proteolytic systemcontaining a reducing agent; (2) observation of the rate of dissolutionin a solution containing reducing agent and a proteinase; (3)measurement of protein leakage from hair samples when exposed for 2hours to a non-proteolytic system containing reducing agent; and (4)measurement of protein leakage from hair samples when exposed for 2hours to a proteolytic system containing reducing agent.

Example 1 Hair Integrity Test of Non-Proteolytic Hair Digests

A solution of dithiothreitol (0.3% DTT, adjusted to pH 9.5 with KOH) wasadded to hair samples (5 mg hair per mL) and incubated for 2 hours. Thesamples were placed in a 37° C. water bath with shaking at 110oscillations per minute (opm) for 2 hours. After 2 hours the sampleswere observed for the extent of visible hair degradation. Samples thathave not been subjected to cosmetic treatments or that have beensubjected to normal cosmetic procedures appear essentially intact.Damage hair samples, however, appear softened and have disintegrated atthis point (FIG. 1). As shown in FIG. 1, the first two samples arenearly destroyed at the end of 2 hours, the third sample is partiallydestroyed, and the two control samples (untreated virgin hair) remainvisually unchanged still have intact-appearing hair strands.

Hair samples that are partially or fully dissolved are identified asdamaged. Alternatively, the samples can be further evaluated using theproteolytic method and rated on a scale of 1 to 8, with 8 beingintact/control hair. An estimate of the state of damaged hair appearancemay be 1-2 (tubes 1 and 2), 4-5 (tube 3) and 7-8 (tubes 4 and 5).

Example 2 Hair Integrity Test of Proteolytic Hair Digests

A solution of dithiothreitol and proteinase K (Sigma P6556), (0.6% DTT,0.25 units/ml proteinase K, adjusted to pH 9.5 with KOH) was added tohair samples (5 mg hair per mL) and incubated for 2 hours. Nonporousintact hair in this solution, at 37oC with shaking, requires about 2hours to dissolve completely, while damaged hair dissolves morequickly—the more damaged the hair the more quickly it dissolves. Thestate of the hair samples is noted at 15-minute intervals, and integrityvalues are assigned depending on the time-point at which the sampledissolves. Integrity values of 1-8 (1=15 min and 8=120 min) wereassigned to correspond to the 8 time-points of dissolution.

As shown in FIG. 2, a series of photographs were taken at 15-minuteintervals to visualize the rate of dissolution. At these 15 minuteintervals the condition of the hair samples in the tube is assessed byvisual inspection and recorded. As part of the assessment, thetechnician observed and recorded the hair solubilization, or simply itsdisappearance. A hair sample that does not significantly dissolve until105-120 minutes is recorded as having maximal integrity (integrity valueof 8). Samples having more than 70% dissolution at 15, 30, 45, 60 and 90minutes are rated as porous and therefore damaged. Referring to FIGS.2A-F, samples 2, 3, 5 and 7 have more than 70% dissolution at 75minutes. Thus samples 2, 3, 5 and 7 are rated as porous and damaged.

A sample that does not reach 70% dissolution by 90 minutes is not ratedas overly porous. For example, samples 1, 4, 6 and 8 have less than 70%dissolution by 90 minutes, and are rated as healthy or intact hairsamples.

Example 3 Measurement of Protein Leakage from Damaged Hair DuringNon-Proteolytic Digestion

For this example, the inventors determined hair porosity by performing aprotein analysis of nonenzymatically (nonproteolytic) digested samplesat the two-hour endpoint to quantify the amount of solubilized hairproteins released from the hair. The proteins measured in the proteinassay may be those solubilized or made “labile” by the cosmetictreatments themselves, as well as proteins released through the actionof the reducing agent (e.g. DTT or DTE) penetrating more readily theporous hair samples.

Using the Bradford protein assay, a standard curve using bovine serumalbumin in concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg/mL isdetermined as a reference. In a 96-well microplate, 2.5 uL of standardsand samples were combined in the wells along with 300 uL of CoomassieBlue reagent (Biorad). The samples were mixed and read after 5-10minutes at 595 mu in a microplate reader. At this sample volume, therewas no interference of dithiothreitol in the assay.

Example 4 Materials and Methods Methylene Blue Staining:

Hair can be identified as porous or nonporous by a previously publishedmethod of staining with methylene blue.^([19]) Use of methylene blue toidentify cosmetically and mechanically damaged hair has also beenpresented by Roe^([20]) and Kuzuhara^([21]). The procedure, althoughsomewhat laborious and impractical in a high through-put laboratory,provides evidence of porosity in cases of permed, bleached, dyed ormechanically damaged hair, and has been routinely used in thislaboratory for this purpose. Four new methods to characterize thecondition of hair samples, the methods for which are presented below,include (1) observation of dissolution of hair in a non-proteolyticsystem containing a reducing agent (e.g., DTT or DTE)^([22,23]); (2)observation of the rate of dissolution in a solution containing reducingagent (e.g., DTT or DTE) and a proteinase suitable for digestion ofkeratin (e.g., proteinase K)^([24]); (3) measurement of protein leakagefrom hair samples when exposed for 2 hours to a non-proteolytic systemcontaining reducing agent (e.g., DTT or DTE); and (4) measurement ofprotein leakage from hair samples when exposed to a proteolytic systemcontaining reducing agent and proteolytic enzyme.

Nonproteolytic Digestion System to Identify Damaged Samples:

The nonproteolytic digestion method consists of 5 mg hair per mL of 0.3%dithiothreitol (Sigma D0632) adjusted to pH 9.5 with KOH. The samplesare placed in a 37° C. water bath with shaking at 110 oscillations perminute (opm) for 2 hours. After 2 hours the samples are observed andthose showing dissolution are noted. Only exceptionally damaged samplesshow such dissolution in this system. Samples that have not beensubjected to cosmetic treatments or that have been subjected to normalcosmetic procedures appear essentially intact. Only about 0.5% ofsamples demonstrate dissolution at 2 hours in the nonproteolyticdigestion. These samples are tested again by the proteolytic method,described below.

Proteolytic (Enzymatic) Digestion System to Identify Damaged Samples:

The proteolytic digestion method consists of 5 mg hair per mL of asolution of 0.6% dithiothreitol at pH 9.5 containing 0.25 unit/mL ofProteinase K (Sigma P6556). Nonporous intact hair in this solution, at37° C. with shaking, requires about 2 hours to dissolve completely,while damaged hair dissolves more quickly—the more damaged the hair themore quickly it dissolves. The state of the hair samples is noted at15-minute intervals, and integrity values are assigned depending on thetime-point at which the sample dissolves. Integrity values of 1-8 (1=15min and 8=120 min) were assigned to correspond to the 8 time-points ofdissolution. Correlation of these values to known severities of cosmetictreatments will be shown in Results.

The effects of cosmetic treatments in these systems were tested withlaboratory-treated hair samples. Each treatment was applied to threesamples. For perming hair, the product used was “Feels So Lively,”Alkaline Perm for Resistant Hair, Zotos International, Inc. Hair wasexposed to the perming solution for the recommended 12 minutes, rinsedunder running tap water, neutralized, rinsed again, and then dried.

To relax hair, the product applied was “Soft and Beautiful No-LyeRelaxer System,” Unilever Corporation. Relaxer components were combinedaccording to kit instruction, applied with an applicator stick, massagedinto the hair, and allowed on the hair for 15 minutes. The sample wasrinsed and neutralizing shampoo applied repeatedly until no pinkindicator color was visible. The samples were rinsed under running tapwater and then allowed to dry.

Bleaching was performed with “L'Oréal Super Blonde Crème LighteningKit,” L'Oréal Group. Solutions were prepared according to kitinstructions, and applied with massage into the hair. The bleach wasallowed on the hair for the recommended 25 minutes, and then rinsedunder running tap water and dried.

In addition, a hair sample with known professional cosmetic treatmentsand untreated control samples were taken from children's hair cuts withno hair treatments were tested.

Measurement of Protein Leakage from Damaged Hair During NonproteolyticDigestion:

The inventors also investigated another means of determining hairporosity by performing a protein analysis of the nonenzymatically(nonproteolytic) digested samples at the 2-hour endpoint to quantify theamount of solubilized hair proteins released from the hair. The proteinsmeasured in the protein assay may be those solubilized or made “labile”by the cosmetic treatments themselves^([26]), as well as proteinsreleased through the action of dithiothreitol penetrating more readilythe porous hair samples.

Using the Bradford protein assay,^([26]) a standard curve using bovineserum albumin in concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg/mL isdetermined as a reference. In a 96-well microplate, 2.5 uL of standardsand samples were combined in the wells along with 300 uL of CoomassieBlue reagent (Biorad). The samples were mixed and read after 5-10minutes at 595 mu in a microplate reader. At this sample volume, therewas no interference of dithiothreitol in the assay.

Wash Methods:

The aqueous wash procedure and application of the wash criterion havebeen extensively presented previously.^([15-18]) Briefly the methodconsists of 10-12 mg hair in 2 mL of an initial non-swelling 15-min dryisopropanol wash in a 37° C. 110-opm shaking water bath, followed bythree 30-min washes and two 60-min washes in 0.01 M phosphate buffer (pH6) containing 0.1% bovine serum albumin (BSA), also in a 37° C. 110-opmshaking water bath. The initial non-swelling isopropanol wash serves toremove drug that may be only on the surface of the hair, prior to addingthe hair-swelling solution that could allow such surface drug topenetrate the hair. The last wash is analyzed for the drug of interest.The wash criterion for cocaine is as follows: if the amount of drug inthe last wash multiplied by 5 and then subtracted from the amount ofdrug in the hair results in a value below the cutoff, this is anindication that the sample may be contaminated or porous. Thiscalculation is essentially a mathematical extrapolation of the washingby another 5 hours, as an indication of whether or not all the drugwould be washed out; this is, of course an overestimate of drug removalsince the wash kinetic curve is never that linear, and if anything errson the side of safety in protecting the subject from being reported aspositive from environmental contamination. For amphetamines, the washcriterion has been determined empirically to be optimal when the lastwash is multiplied by 3.5 and the result subtracted from the amount ofdrug in the hair.^([15,17])

The ethanolic wash method presented herein, first noted in 1992 as analternative wash for porous samples,^([27]) consists of three 30-min andtwo 60-min washes in 90% ethanol, also performed in a 37° C. 110-opmshaking water bath. The last wash is analyzed for the drug of interestby first acidifying the sample (to prevent volatilization of theamphetamines) and then evaporating the ethanol, followed byreconstituting the sample in 0.01 M phosphate buffer (pH 6) containing0.1% BSA.

Wash samples are analyzed either by LC/MS/MS, by the same procedures asdescribed below for extracts of hair samples, or by quantitative enzymeimmunoassays developed in this laboratory for analysis of hair washes.

In Vitro Contamination of Hair Samples with Cocaine or Methamphetamine:

To test the need for extended exposure to aqueous or swelling solventsfor effective washing, samples from ponytails were soaked in 10 ugcocaine/mL water for 1 hour, rinsed and dried overnight. In addition towashing by the extended washing methods described above, short exposuresto two different nonswelling solvents, dichloromethane and methanol,were also tested. In each case, the 11-12 mg of the hair samples werewashed twice with 2 mL of the solvents for 2 minutes each with gentlevortexing, and the washes and washed hair analyzed for cocaine andamphetamines. Total drug on the hair was calculated by addition of thedrug in the washes and in the hair.

Confirmation Procedures—Cocaine:

The washed hair was enzymatically digested at pH 5.5 as previouslydescribed.^([14,17,18]) Digest supernantants were extracted usingIsolute SPE columns. Eluted samples were dried and 25 uL ofN,N-dimethylformamide dipropyl acetal was added, with heating at 120° C.for 5 minutes, to effect the derivatization of benzoylecgonine, toensure separation of norcocaine from the benzoylecgonine.

LC/MS/MS analysis of cocaine and metabolites was performed on a PerkinElmer Sciex triple quadrupole API 2000 (Thornhill, On, Canada) MS,operating in the positive chemical ionization mode, linked to two PerkinElmer Series 200 micropumps and a Perkin Elmer Series 200 autosampler.Mass resolution on Q1 and Q3 were set to unit resolution. Interfaceheater was ON, Turbo ion spray voltage was 5500V; Turbo gas temperaturewas 350° C. to 400° C.; in Positive Reaction Mode. The mobile phase was33% acetonitrile and 0.1% formic acid and 67% water with 0.1% formicacid. Isocratic mode was used. The HPLC column was a Keystone ScientificBETASIL C8, 2 mm×50 mm, 5 μm particle size; a flow rate of 300 μL/minutewas used. For LC, the autosampler with two micropumps used a 5 μm sampleloop volume, 10 μL injection volume, 500 μL flush volume, and a flushsolvent of acetonitrile:water: formic acid (30:70:1). The transitionsfor cocaine and its internal standard cocaine-d3 were m/z 304.1>182.1and m/z 301.1>185.1, respectively; for derivatized benzoylecgonine andits d3-internal standard, m/z 332.1>210 and m/z 335.1>213; forcocaethylene and its d3-internal standard, m/z 318.1>196 and 321.1>199;for norcocaine and its d3-internal standard, m/z 290.1>168 and m/z293.1>171.

Confirmation Procedures—Methamphetamine:

The washed hair was enzymatically digested at pH 6.65 as previouslydescribed.^([15]) Digest supernatants were extracted with aliquid/liquid extraction method. LC/MS/MS analysis of amphetamines wasperformed on a Perkin Elmer Sciex triple quadrupole API 2000 (Thornhill,On, Canada) MS, operating in the positive chemical ionization mode,linked to two Perkin Elmer Series 200 micropumps and a Perkin ElmerSeries 200 autosampler. Mass resolution on Q1 and Q3 were set to unitresolution. Interface heater was ON, Turbo ion spray voltage was 5500V;Turbo gas temperature was 350° C. to 400° C.; in Positive Reaction Mode.The mobile phase was 11% acetonitrile with 0.1% formic acid and 89%water with 0.1% formic acid. Isocratic mode was used. The HPLC columnwas a Keystone Scientific BETASIL C8, 2 mm×50 mm, 5 μm particle size; aflow rate of 300 μL/minute was used. For LC, the autosampler with twomicropumps used a 5 μL sample loop volume, 10 μL injection volume, 500μL flush volume, and a flush solvent of acetonitrile:water: formic acid(30:70:1).

The target ions for methamphetamine and its internal standardmethamphetamine-d11 were m/z 150<91 and m/z 161>96, respectively; foramphetamine and its internal standard amphetamine-D8, m/z 136>91 and m/z144>96; for MDMA and its internal standard MDMA-D5, m/z 194>135 and m/z199>136; for MDEA and its internal standard MDEA-D6, m/z 194>163 and m/z199>166. Specificity studies ruled out interference from ephedrine,fenfluramine, pseudoephedrine, phentermine and phenylpropanolamine.

Results: Hair Integrity Testing Cosmetic Treatments and Rate of SampleDissolution in Proteolytic Digestion

As described in Methods, previously untreated hair samples werecosmetically treated with bleach, perm, and relaxer, and then subjectedto the timed proteolytic digestion. Also, some hair samples known tohave no prior cosmetic treatments and one with known professionalcosmetic treatments were tested. As shown in Table 1, the untreatedcontrol samples dissolved at the 105-120-minute timing, while treatedsamples dissolve earlier depending on the severity of the treatments.Normal perm or bleach treatments, following the vendors' instructions,resulted in shorter digestion times than the controls, usually in therange of 60-90 minutes, with assigned Integrity Values of 4-6, asdescribed in Methods. When excessive bleaching was intentionallyperformed, the samples digested more quickly with each additionaltreatment, with 3 bleachings dissolving in 30 minutes (Integrity Valueof 2). The salon-dyed hair, from a female who has the roots dyedapproximately monthly, dissolved in 90 minutes. Permanent dyeing of hairusually involves some mild bleaching along with the dye, and it can beassumed that it is the bleach that causes the hair damage.

TABLE 1 Control and Laboratory Cosmetically Treated Samples inProteolytic Digestion Time for Hair to Integrity Type of Sample Dissolve(minutes) Value Control: Haircut, male 120 8 Control: Ponytail-1, female105 7 Control: Ponytail-2, female 120 8 Female -white hair, 90 6routinely salon dyed Permed #1 75 5 Permed #2 90 6 Permed #3 75 5Bleached #1 60 4 Bleached #2 60 4 Bleached #3 60 4 Bleach #3 twice(excess) 45 3 Bleach #3 times (extreme) 30 2 Relaxed #1 Did not dissolvePorous* Relaxed #2 Did not dissolve Porous* Relaxed #3 Did not dissolvePorous* *See text for experiment performed with relaxed hair todemonstrate porosity.

Samples with integrity values of 2 or less are excessively damagedsamples, as shown with the triple-bleached sample, and may be reportedas invalid or unsuitable samples for testing for drug use, since suchdamage can remove any drug deposited due to ingestion during thetreatment itself and in subsequent repeated shampooing, resulting in afalse negative report of drug ingestion. Integrity values from 3 to 6indicate sufficient porosity to influence results of aqueous washing ofthe hair sample, since the increased porosity will allow drug depositedfrom ingestion to diffuse out of the hair in an aqueous hair-swellingwash buffer. Integrity values of 7 indicate slight porosity, and 8indicates an intact sample.

Table 1 also shows a major exception to the rapid enzymatic digestion ofcosmetically treated hair—that of hair that has been relaxed orstraightened using very highly basic crème applied to the hair withcombing to straighten the strands (e.g., SoftSheen Optimum Care No-LyeRelaxer). With this treatment the hair does not dissolve at all at 2hours of enzymatic digestion (in fact, a relaxed hair does not dissolvein 24 hours). This characteristic of relaxed hair actually provides thetest for identifying relaxed hair, since normal hair dissolves by 120minutes. In order to determine whether hair is rendered porous by therelaxing treatment, we selected three cocaine-positive samplespreviously shown to be intact, nonporous samples by the fact that thelast wash contained very little cocaine, i.e., the drug present in thehair from ingestion did not diffuse into the aqueous wash solution(Table 2). These samples were relaxed as described in Methods, and therelaxed samples subjected to the aqueous wash procedure. It can be seenthat more drug diffused into the washes after the samples had beenrelaxed. High cocaine values in the wash or failed wash criterionnormally would indicate that the samples were contaminated or porous,but in this case we had selected samples that were not contaminated, sothe failed washes after aqueous washing indicate porosity. Thus, if asample does not dissolve in the proteolytic digestion, this indicatesthe hair is relaxed and porous.

TABLE 2 Effect of Relaxing on Hair Porosity: NancontaminatedCocaine-Positive Hair Samples Before and After Relaxing Treatment IntactSamples before Relaxing Samples after Relaxing Last Last COC BE WashWash COC BE Wash Wash Sample ng/10 mg hair % BE Criterion Interpretationng/10 mg hair % BE Criterion Interpretation 1 160 21.9 7.7 13.7 121.5Positive 66 7.9 16.4 12.0 −16 Contaminated or Porous 2 44.8 4.8 1.5 10.837.1 Positive 47.7 4.8 8.2 10.1 6.7 Positive 3 499.9 60.7 1.4 12.1 492.9Positive 340.1 35.1 108.6 10.3 −202.9 Contaminated or Porous

Dissolution of Hair in Nonproteolytic Digestion System

Evidence of resistance of most hair samples to digestion in thenonproteolytic system is that over 99% of many thousands of samples havenot shown dissolution at the 2-hour point. Examples of samples that doshow disintegration at this time point and were also tested with theproteolytic system to confirm the initial result are shown in Table 3.The proteolytic test, which provides a clearer estimation of porosity,is always performed on those samples identified in the nonproteolyticsystem to confirm the first visual identification of an extremelydamaged hair. Samples that dissolve in 30 minutes or less in theproteolytic system (Integrity Values of 1 or 2) are severely damaged, aswe showed in our testing of laboratory-treated samples.

TABLE 3 Proteolytic Test of Samples that Dissolved at 2-hours inNonproteolytic Digestion Time for Hair to Integrity Sample descriptionDissolve (minutes) Value Blond, straight 45 3 Black, straight, dyed 30 2Brown, straight 45 3 Brown, straight 30 2 Blond, straight 45 3 Brown,straight 30 2Measurement of Protein Leakage from Damaged Hair During NonproteolyticDigestion

The correlation of results of the proteolytic digestion method forporosity and the protein assay performed on nonproteolytic digests areshown in FIG. 5. Some of the samples shown in the correlation weretreated in the laboratory, using commercial perming or bleaching kits,and others were unknown samples characterized by the porosity testing bydigestion rate. Relaxed hair is again an exception in that protein isnot released with these samples. Therefore, the integrity testconsisting of enzymatic digestion rates appears to be the only methodavailable at this time for identifying relaxed hair. Another shortcomingof this protein assay to measure severity of damage is that veryseverely damaged samples may leak so much protein that they exceed therange of the assay, causing precipitation and producing an artificiallylow reading. The artifact can be managed by observing the precipitate inthe wells and performing multiple greater dilutions of the samples.

In Vitro Contamination Studies

Extended Washing with 90% Ethanol

In a preliminary test of 90% ethanol washing, hair samples taken fromponytails purchased from a salon were soaked for 70 minutes in a watersolution of 10 ug/mL cocaine (COC) and methamphetamine (METH), a seriouschallenge to a washing procedure that needs to identify contamination atthe 5 ng/10 mg hair level. The samples were removed after soaking,rinsed under running tap water, and allowed to dry overnight. Sampleswere weighed and then washed with the 90% ethanol procedure, withresults shown in Tables 4 (cocaine) and 5 (methamphetamine). The largeamount of drugs remaining in the hair after washing reflects the highlyconcentrated soaking conditions. Application of the wash criterion,however, clearly identified all samples as negative or contaminated, asseen by the negative values after its application.

TABLE 4 90% Ethanol Washing of Samples Soaked in 10 ug/mL Cocaine (A)Cocaine in Hair (B) Cocaine Wash After in Last Criterion Washing Wash(A) − 5x(B) Sample # ng/10 mg hair Result* 1 43.1 26.6 −89.9Negative/Contam 2 93.9 40 −106.1 Negative/Contam 3 47.8 31.2 −108.2Negative/Contam 4 36 17.3 −50.5 Negative/Contam 5 77.7 18.7 −15.8Negative/Contam 6 54.8 18.7 −38.7 Negative/Contam 7 53 29 −92Negative/Contam 8 42.2 25.1 −83.3 Negative/Contam 9 78 34 −92Negative/Contam

TABLE 5 90% Ethanol Washing of Samples soaked in 10 ug/mLMethamphetamine (A) Meth in Hair (B) Meth Wash After in Last CriterionWashing Wash (A) − 3.5x(B) Sample # ng/10 mg hair Result* 1 77.8 53.5−109.4 Negative/Contam 2 123.5 51.6 −57.1 Negative/Contam 3 74 81.8−212.3 Negative/Contam 4 121.3 53.3 −65.2 Negative/Contam 5 50.6 24.6−35.5 Negative/Contam 6 103.9 34.9 −18.2 Negative/Contam 7 74.3 25.8 −16Negative/Contam 8 242.9 81.8 −43.4 Negative/ContamShort Washes with Alternate Solvents

The inventors were interested in non-hair-swelling solvents for possiblewashing of porous hair, and therefore also tried short exposures to twodifferent solvents, dichloromethane and methanol (Table 6). For all butone of the dichloromethane-washed samples, less than 2.5% of thecontamination was removed; in one case 8.7% was removed by the washing.In the case of methanol washing, 2.2 to 12.9% of the COC was removed,with one case being above 10%. These results support the need forlengthy washing in at least partially hair-swelling solvents along withan indicator or criterion to estimate the effectiveness of thedecontamination process.

TABLE 6 Dicchloromethane and Methanol Washing of Cocaine-Soaked HairSamples Dichloromethane Washing Methanol Washing 1^(st) 2^(nd) Washed1^(st) 2^(nd) Washed Sample wash wash Hair % of Total Drug wash washHair % of Total Drug # ng cocaine/10 mg hair removed ng cocaine/10 mghair removed 1 0.7 0.3 114.5 0.9 3.2 1.7 135.7 3.5 2 3.5 2.8 65.8 3.78.9 3.2 81.7 12.9 3 1.3 0.8 96.5 2.1 5.8 2.5 78.7 9.5 4 2.7 1.7 173.52.5 2.4 1.9 191.6 2.2 5 1.1 1.2 113.1 2.0 5.4 2.5 92.1 7.3 6 0 0 135.20.0 1.9 2.1 118.8 3.3Contaminated Porous and Nonporous Hair with Extended Aqueous and 90%Ethanol Washing

Since the porosity of the samples in Tables 4 and 5 had not beencharacterized, another set of samples was permed to make the samplesporous and determine whether the 90% ethanol wash would identify knowncontaminated porous as well as nonporous samples. Permed and unpermedsamples were soaked in 1000 ng/ml of COC and METH for one hour at roomtemperature. Both aqueous and ethanolic washing results for COC-soakedpermed and unpermed hair samples are shown in Table 7.

TABLE 7 Aqueous and 90% Ethanol Washing of Porous and Nonporous Samplessoaked in 1000 ng/mL Cocaine* (A) Cocaine in Hair (B) Cocaine Wash Afteris Last Criterion Washing Wash (A) − 5x(B) Sample # ng/10 mg hairResult* Aqueous Buffer Washing 1 8.8 1.9 −0.9 Negative/Contam 2 18.2 7.2−18.1 Negative/Contam 3 15.4 3.8 −3.8 Negative/Contam 4 3.0 0 NANegative 5 2.6 0.4 NA Negative 1 Permed 1.3 2.8 NA Negative 2 Permed 2.04.1 NA Negative 3 Permed 4.0 4.2 NA Negative 4 Permed 4.4 3.6 NANegative 5 Permed 0.7 0.9 NA Negative 90% Ethanol Washing 1 15.3 4.8−8.7 Negative/Contam 2 18.8 11.6 −39.2 Negative/Contam 3 21.5 7.9 −18.1Negative/Contam 4 5.5 0.9 1.0 Negative/Contam 5 3.7 0.7 NA Negative 1Permed 103.9 26.7 −29.6 Negative/Contam 2 Permed 97.0 28 −43.0Negative/Contam 3 Permed 68.1 19.8 −30.9 Negative/Contam 4 Permed 71.814.0 1.8 Negative/Contam 5 Permed 21.6 6.5 −10.9 Negative/Contam *TotalCocaine on unpermed hair ranged from 10.2-91.9 ng/10 mg hair; totalcocaine on permed hair ranged from 73.9-221.1 ng/10 mg hair. Cutoff forcocaine in hair is 5 ng/10 mg hair.

The total COC in the unpermed samples ranged from 10.2 to 91.9 ng/10 mghair; in the permed samples, the total COC taken up by the hair was73.9-221.1 ng/10 mg hair. Although, as expected, the uptake in thepermed samples was greater than in the unpermed, both permed andunpermed samples were either negative after washing (under cutoff) orfailed the wash criterion, indicating contamination. It is generally thecase with contamination of porous samples that “what goes in easily alsocomes out easily.” Prior cosmetic histories of the ponytails receivedfrom the salons was not known, so it is possible some of the hair thatwe permed may already have had some prior treatment to cause someporosity, accounting for the wide range of uptake among the unpermedsamples. Also, older hair from ponytails is often porous due tomechanical damage over time (For in vitro experimenters, this is a noteof caution that ponytail hair may not be representative of the 0-3.9 cmproximal samples usually tested in workplace testing.) With the aqueousbuffer washing, 3 unpermed samples were above the cutoff after washingand were identified as negative or contaminated by application of thewash criterion. Two unpermed and all of the permed samples were negativeby the cutoff after washing and did not require application of the washcriterion. With ethanolic washing, one unpermed sample fell below thecutoff, and 4 unpermed and all 5 permed samples fell above the cutoffbut were identified as negative or contaminated after application of thewash criterion, thus demonstrating the effectiveness of the 90% ethanolwash and use of the wash criterion with this wash solvent to identifyCOC contamination.

With the METH-soaked samples, the range of METH taken up by the unpermedhair samples was 4.6-66.0 ng/10 mg hair (average=26), and for the permedsamples was 33.3 to 95.6 ng/10 mg hair (average=52) (Table 8). Allexcept one unpermed sample fell below the cutoff after aqueous washing,and one fell above the cutoff and was identified ascontaminated/negative by the wash criterion. After ethanolic washing,three unpermed samples fell below the cutoff after washing and theremainder of the samples were identified as contaminated/negative by thewash criterion. Also, none of these samples contained the metabolite(AMP). This supports the conclusion that the ethanolic washing and washcriterion identifies contamination for both intact and porous hair.

TABLE 8 Aqueous and 90% Ethanol Washing of Nonporous and Porous HairSamples soaked in 1000 ng/mL Methamphetamine* (A) Meth in Hair (B) MethWash After in Last Criterion Washing Wash (A) − 3.5x(B) Sample # ng/10mg hair Result* Aqueous Buffer Washing 1 0 0 NA Negative 2 1.3 0.5 NANegative 3 8.8 4.6 −7.3 Negative/Contam 4 1.4 0.8 NA Negative 5 0 0 NANegative 1 Permed 0 0 NA Negative 2 Permed 0 0 NA Negative 3 Permed 0 0NA Negative 4 Permed 0 0 NA Negative 5 Permed 0 0 NA Negative 90%Ethanol Washing 1 4.5 3.0 NA Negative 2 7.3 0.9 0.7 Negative/Contam 35.0 8.6 −25.1 Negative/Contam 4 4.2 0.4 NA Negative 5 3.7 4.9 NANegative 1 Permed 16.1 5.3 −2.5 Negative/Contam 2 Permed 17.6 6.5 −5.2Negative/Contam 3 Permed 29.1 7.9 1.5 Negative/Contam 4 Permed 26.2 8.4−3.2 Negative/Contam 5 Permed 5.5 3.5 −6.8 Negative/Contam *Total Methon unpermed hair ranged from 4.6-66.0 ng/10 mg hair (average = 26);total meth on permed hair ranged from 33.3-95.6 ng/10 mg hair (average =52). Cutoff for methamphetamine is 5 ng/10 mg hair

Washing Studies on Cocaine and Methamphetamine-Positive Hair SamplesCocaine

A demonstration of ethanolic washing for previously identifiedCOC-positive hair compared aqueous washing of positive COC hair sampleswith ethanolic washing (Table 9). These samples did not fail the washcriterion using the aqueous solvent, and the intent was to assessdifferences in results with aqueous vs ethanolic washing. With thesesamples, which were only moderately porous or nonporous (IntegrityValues of 5-7), both methods produced essentially similar results, andall samples were positive by both methods, passing the wash criterion,with COC above the cutoff and BE>5% of COC.

TABLE 9 Aqueous and 90% Ethanol Washing of Moderately and MinimaillyPorous hair Washes Hair Results Washing Resltus COC BE Last Wash SampleIntegrity Value Wash Type ng/10 mg hair % BE (ng COC/10 mg hair) WashCriterion* Pass/Fail 1 5 Ethanolic 25.6 1.4 5.5 1.8 16.6 P Aqueous 24.51.3 5.3 1.0 19.5 P 2 6 Ethanolic 587.9 92.9 15.8 47.2 351.9 P Aqueous628.7 80.7 12.8 32.4 466.7 P 3 7 Ethanolic 33.4 1.3 5.7 1.3 26.9 PAqueous 34.1 1.7 5.0 3.3 17.6 P 4 7 Ethanolic 1274.3 161.2 12.7 107.1738.8 P Aqueous 1386.7 222.5 16.0 70.1 1036.2 P 5 6 Ethanolic 38.2 3.59.2 0.7 34.7 P Aqueous 34.8 2.3 6.6 0 34.8 P 6 7 Ethanolic 23.3 5.3 22.70 23.3 P Aqueous 33.4 5.5 16.5 0 33.4 P 7 6 Ethanolic 12.8 1.3 10.2 012.8 P Aqueous 15.6 1.4 9.0 0 15.6 P

Next, fourteen hair samples with aqueous wash results suggestingcontamination or porosity were tested by the digestion-rate integritytest, with results showing moderate damage or porosity (Integrity Valuesof 4-6), and one relaxed sample (Table 10). These samples were thenwashed with the 90% ethanol wash. Results of the aqueous and ethanolicwashes and application of the wash criterion in each case are shown inTable 7. In all of the cases in the table, the samples passed the washcriterion after ethanolic washing, and were positive with COC above thecutoff and BE>5% of COC. In some cases (e.g., #3, 4, 5, 6, 11), theaqueous wash appeared to preferentially extract BE, as suggested by thelower BE in the aqueous washed sample.

TABLE 10 Aqueous and 90% Ethanol Washing of Porous Cocaine-ContainingHair Washed Hair Results Washing Results COC BE Last Wash SampleIntegrity Value Wash Type ng/10 mg hair % BE (ng COC/10 mg hair) WashCriterion* Pass/Fail 1 6 Ethanolic 106.7 16.7 15.7 3.6 88.7 P Aqueous 619.8 16.1 26.5 −71.5 F 2 5 Ethanolic 97 8.5 8.8 5.3 70.5 P Aqueous 106.98.3 7.8 29.8 −42.1 F 3 5 Ethanolic 30.8 5.7 18.5 3.5 13.3 P Aqueous 21.62.8 13.0 6.8 −9.9 F 4 5 Ethanolic 99.6 8.6 8.6 15.1 24.1 P Aqueous 76.62 2.6 17.7 −11.9 F 5 6 Ethanolic 27.3 5.9 21.6 1.1 21.8 P Aqueous 26.64.3 16.2 5.2 0.6 F 6 5 Ethanolic 34.1 12.2 35.8 2 24.1 P Aqueous 38 9.324.5 12.9 −26.5 F 7 5 Ethanolic 600.9 61.3 10.2 46.2 369.9 P Aqueous368.6 62.1 16.8 81.3 −37.9 F 8 4 Ethanolic 155.4 32.0 20.5 23.3 38.9 PAqueous 158.5 28.1 17.7 38.2 −32.5 F 9 6 Ethanolic 12.6 2.4 19.0 1.1 7.1P Aqueous 21.8 3.5 16.0 20 −78.2 F 10 6 Ethanolic 11.6 2.6 22.4 1.4 5.1P Aqueous 18.6 4.5 24.1 9.8 −30.4 F 11 5 Ethanolic 13.9 3.5 25.1 1.3 7.4P Aqueous 10.8 2.2 20.3 2.1 0.3 F 12 5 Ethanolic 14.2 0.8 5.6 1.5 6.7 PAqueous 11.1 0.7 6.3 2.7 −2.4 F 13 Relaxed Ethanolic 616.7 40.7 6.6 76.8232.7 P Aqueous 736.0 41.0 5.6 382.8 −1178 F 14 6 Ethanolic 12.2 2.218.0 0.9 7.7 P Aqueous 11.3 1.8 15.9 3.0 −3.7 F

Table 11 shows samples that failed the wash criterion for contaminationafter washing with either aqueous or ethanolic solvents. These samplesare not reported positive; they may be reported as follows: “The hairsample submitted is an unsuitable specimen that appears to be heavilycontaminated with drug beyond that which can be removed or accountedfor.”

TABLE 11 Cocaine-containing samples falling both Aqueous and 90% EthanolWashing and not reported as positive Washed Hair Result Washing ResultsCOC BE Last Wash Sample Integrity Value Wash Type ng/10 mg hair % BE (ngCOC/10 mg hair) Wash Criterion* Pass/Fail 1 4 Ethanolic 25 2.7 10.8 4.90.5 F Aqueous 30.2 0.9 3.0 14.5 −57.7 F 2 Relaxed Ethanolic 24.2 2.510.3 51.1 −231.3 F Aqueous 17.8 0.6 3.4 62.9 −296.7 F 3 6 Ethanolic 17.41.8 10.3 4.3 −4.1 F Aqueous 14.7 1.2 8.2 4.3 −6.8 F 4 Relaxed Ethanolic29.5 1.6 5.4 8.1 −1.1 F Aqueous 30.5 0.6 2.0 45.4 −196.5 F 5 7 Ethanolic84.2 26.1 31.0 54.2 −186.8 F Aqueous 273.0 41 15.0 267.6 −1065 F 6 8Ethanolic 49.6 3.8 7.7 25 −75.4 F Aqueous 60.2 3.3 5.5 15 −14.8 F 7 8Ethanolic 15.9 4.0 25.2 10.6 −37.1 F Aqueous 18.1 4.3 23.8 8.3 −23.4 F 84 Ethanolic 19.9 2.1 10.6 4.6 −3.1 F Aqueous 22.0 2.3 10.5 8.9 −22.5 F

Methamphetamine

Some moderately porous samples that previously tested positive for METHafter aqueous washing were tested for integrity and washed with theethanolic method (Table 12). The aqueous-washed hair had lower METHvalues in the case of #s 1 and 5, but all samples were positive by boththe aqueous and ethanolic wash methods. This supports the validity ofwashing with the ethanolic method.

TABLE 12 Aqueous and 90% Ethanol Washing of Moderately PorousMethamaphetamine-Positive Hair Hair after Washing Wash Content IntegrityWash METH AMP METH AMP Wash WC Sample # Value Method ng/10 mg hair % AMPng/10 mg hair % AMP Criterion Pass/Fail 1 4 Ethanol 110.4 8.7 7.9 2.90.2 NA 100.3 P Aqueous 70.8 4.9 6.9 14.9 1.7 11.4  18.7 P 2 6 Ethanol68.3 4.1 6.0 2.3 0.1 NA 60.3 P Aqueous 78 4.5 5.8 7.6 0.5 6.6 51.4 P 3 5Ethanol 157.2 19.8 12.6 3.2 0.3 9.4 146.0 P Aqueous 154.6 16.9 10.9 28.94.2 14.5  53.5 P 4 6 Ethanol 88.9 4.6 5.2 0.3 0 NA 67.9 P Aqueous 65.12.8 43 5.5 0.3 5.5 45.9 P 5 5 Ethanol 142.1 9.4 6.6 4.7 0.3 6.4 125.7 PAqueous 115.5 7.3 6.3 14.5 1.3 9.0 64.8 P

Another 20 twenty samples were tested for integrity when the aqueouswash criterion result indicated either contamination or porosity. Theresults of aqueous and ethanolic washing, shown in Table 13, show thatin all these cases of increased porosity the ethanolic washing producedan acceptable Wash Criterion. Comparing results of ethanolic washingversus aqueous washing can show a variety of effects in amounts of drugin the washes and amounts remaining in hair. This is likely due tocombined effects of true contamination, porosity and amount of ingesteddose.

TABLE 13 Aqueous and 90% Ethanol Washing of Methamphetamine-ContainingHair Samples Washing Results Hair after Washing Wash Intergrity WashMETH AMP % Last Wash Wash Criterion Sample # Value Method ng/10 mg hairAMP ng METH/10 mg hair Criterion Pass/Fail 1 3 Ethanolic 16.8 0.5 3.0 29.8 P Aqueous 16.5 0.4 2.4 3.4 4.6 F 2 4 Ethanolic 10.4 0.8 7.7 0.5 8.65P Aqueous 6.6 3.5 7.4 1.2 2.6 F 3 4 Ethanolic 18.5 1.4 10.4 0.5 11.75 PAqueous 9.6 1.1 11.5 5.8 −10.7 F 4 5 Ethanolic 6.8 0.5 7.4 0 6.8 PAqueous 7.5 0.5 6.7 2.5 −1.25 F 5 5 Ethanolic 8.5 0.5 5.9 0.4 7.1 PAqueous 8.2 3.5 6.1 1.3 3.65 F 6 6 Ethanolic 10.6 1 9.4 0.9 7.45 PAqueous 6.4 0.5 7.8 0.8 3.6 F 7 6 Ethanolic 39.9 7.2 18.0 4.6 23.8 PAqueous 34.9 6 17.2 11.5 −5.35 F 8 6 Ethanolic 14.9 2.7 18.1 0.9 11.75 PAqueous 9.4 1.4 14.9 3.3 −2.15 F 9 4 Ethanolic 83.2 6.6 7.9 4 69.2 PAqueous 24.1 1.6 6.6 11.8 −17.2 F 10 6 Ethanolic 19.5 1 5.1 1.4 14.6 PAqueous 8.4 0.6 7.1 3.1 −2.45 F 11 6 Ethanolic 32.6 2.8 8.6 3.7 19.65 PAqueous 26.2 2 7.6 7.2 1 F 12 4 Ethanolic 10.5 1.3 12.4 0.4 9.1 PAqueous 5.7 0.36 6.3 2 −1.3 F 13 5 Ethanolic 9.5 0.6 6.3 0 9.5 P Aqueous7.5 0.5 6.7 1.6 1.9 F 14 4 Ethanolic 6.7 0.9 13.4 0 6.7 P Aqueous 5.30.7 13.2 1.2 1.1 F 15 4 Ethanolic 62.9 3.1 4.9 6.3 40.85 P Aqueous 40.91.5 3.7 12.1 −1.45 F 16 4 Ethanolic 19.6 1.3 6.6 2.1 12.25 P Aqueous24.2 1.5 6.2 7.4 −1.7 F 17 4 Ethanolic 21.8 3.4 15.6 2.4 13.4 P Aqueous19.2 2.9 15.1 4.5 3.45 F 18 6 Ethanolic 12.6 1.3 10.3 0.6 10.5 P Aqueous8.7 0.8 9.2 1.2 4.5 F 19 4 Ethanolic 19.2 1.3 6.8 2 12.2 P Aqueous 8.70.6 6.9 4.3 −6.35 F 20 4 Ethanolic 25.2 2.7 10.7 2.4 16.8 P Aqueous 10.51 9.5 6.6 −12.6 F

In all cases in Table 13, the presence of the metabolite amphetamine wasshown, with ratios from 3.0 to 18.1% of METH in the ethanolic washedhair. The presence of AMP in the hair as a metabolite is strong evidenceof METH ingestion. One report with controlled doses of METH stated arange in a set of 7 subjects from 7 to 37% of methamphetamine^([26]).The same situation pertains here as in workplace testing for otheranalytes where prescription drugs can confound testing results, in whichcase the subject can produce proof of a prescription. In the case ofMETH/AMP, if a subject can demonstrate prescribed use of selegiline oramphetamine, this may affect the MRO's disposition of the findings.Also, in the case of selegiline, the metabolic products are 1-METH and1-AMP which can be distinguished from the d forms upon request.

With porous hair, especially in the case of METH where the analyte isvery readily extracted by water, the last wash is in fact a partialextraction of the hair, with the evidence for that being that themetabolite ratio in the wash is similar to that in the digest of thehair after washing. Thus, although washing is critical to determinationof contamination, analysis of the wash for both the parent and itsmetabolite can also indicate whether drug from ingestion is entering thewash medium due to porosity of the hair. In cases of METH contamination,there should be little or no AMP present, as was seen in our in vitrocontamination experiments above. In all of the results of 90% ethanolwashing of 12 samples in Table 13, some of which fail the wash criterionand others pass, the amp/meth as percent of the washes was greater than70% of that in the digested hair samples. The use of ratios of AMP inMETH-containing samples and washes is an ongoing investigation that mayprovide an additional criterion to distinguish ingestion fromcontamination.

Table 13 presents more METH-containing samples, with their IntegrityValues, that fail the wash criterion with both aqueous and ethanolicwashing. In cases of severe porosity (Samples 1 and 2, Integrity valuesof 3 and 4) it is not certain whether the failed wash criterion is dueto extreme porosity or contamination. Both samples were above cutoff andthe AMP:METH ratios may indicate ingestion. In nonporous hair (Samples3-8, integrity values of 6-8), the failed wash criterion signals areport of contamination. However, the presence of metabolite in thesesamples suggests a contaminated user.

Application of extended aqueous washing of hair has been shown to beboth essential and effective in protecting against false positives dueto external contamination, even in extreme laboratory simulations. Theaqueous wash method described here has been validated by anotherlaboratory as being successful in identifying contaminatedsamples.^([29]) In that exercise, a vigorous attempt to simulate apossible environmental contamination with COC included rubbing COC onhair followed by storage and 3 shampooings per week for a month. Sampleswere tested through-out the month-long experiment. The paper reportedthat using this wash procedure, all samples were identified ascontaminated using a 5 ng/10 mg hair cutoff, a 0.05 BE/COC ratio, andthe wash criterion. The method was again validated in a report to theU.S. Department of Justice, using similar contamination methods and thesame criteria.^([30]) This report also found that a wash method usingshort exposures to non-swelling agents, in this case methanol,^([31])was unsuccessful in identifying the samples as contaminated. This isconsistent with the present results with COC-contaminated samples whereshort methanol and dichloromethane washings were inadequate todecontaminate the samples or identify them as contaminated. It should benoted as well that drug contamination is not ubiquitous in theenvironment, as evidenced by the fact that the great majority of samplesare negative in the screening assay which is performed on unwashedsamples. In fact, most samples that are contaminated are the result ofusers contaminating their own hair during use. On the other hand, thislaboratory has reported results of clear-cut contamination, such as acase of a chest hair sample that was well above cutoff for METH butfailed the wash criterion and lacked the metabolite AMP. The donor wasfound to be a dealer who kept drug in his shirt pocket.

Numerous reports of effects of cosmetic treatments on hair drug contenthave been reported, but, to date, with no method of detecting damagedhair during the analysis, leaving a gap as to the reliability ofnegative results, especially in light of increasing public awareness ofmethods for evasion of detection. Performing a search online with thewords “beat hair test” produces 85,000,000 hits. Many of these arewishful thinking, but there are some that suggest excessive bleachingand re-dyeing of the hair, which can produce negative results even atthe screening level, without any washing considerations. With the testsdescribed herein, these excessively damaged samples can be detected andthen re-tested for confirmation of integrity status by either theproteolytic digestion rate method or the protein assay method.

The degrees of porosity of hair samples fall on a continuum from minormechanical damage to normal cosmetic treatments to extreme treatments,possibly with an intent to evade detection of use. If a sample is moremoderately damaged, it will not be a false negative in the screeningassay, but has the potential of falling below the cutoff after extensivewashing, or of failing the wash criterion. The possibility of washingout drug from a damaged/porous sample does not justify eliminatingwashing, as washing is necessary to ensure against false positives dueto external contamination as well as to remove drug deposited by sweat.Accumulation of drug from sweat on the hair of a person who does notregularly shampoo can seriously overestimate the dose and cause a lightuser to be above the cutoff whereas the same level of use by a subjectwith daily shampoo habits may fall below the cutoff. Extended washingmitigates such effects. What the occurrence of porous/damaged samplesdoes require is a method to determine the reason for a failed washcriterion or a discrepancy between a screening result and the drug inthe washed hair. In our experience, about 10% of COC-containing and 20%of METH samples fail the aqueous wash criterion, requiring an integritytest, re-washing with 90% ethanol and re-evaluation using cutoff,metabolites, and wash criterion. If a confirmation result after aqueouswashing is low and inconsistent with a strong screening result, thesample needs to be subjected to an integrity test to determine theintactness/porosity of the hair. This information is then used to selecta wash procedure appropriate for the state of the hair. The same appliesto a sample that fails the wash criterion. A wash procedure that appearsto be suitable for porous hair is the 9:1 ethanol:water washing over 3.5hours, presented in this paper. It is suggested that the criticalcomponents of the wash procedure are the presence of enough water (orother sufficiently swelling solvent) to (1) enter the variously damagedhair samples, and (2) effect the solubilization and diffusion of theanalyte out of the hair. The 90% ethanol procedure was shown to beeffective in identifying in vitro contaminated porous and nonporoussamples, and in identifying user samples where metabolites or prior datasupported the interpretation of a positive sample due to ingested drug.

Example 5 Hair Integrity Test of Proteolytic Digests of Hair Samples:KOH Treatment

FIG. 3 is a photograph demonstrating a hair sample subjected to KOHtreatment prior to treatment using a proteolytic digestion (left-handtube) and a control sample subjected to treatment using a proteolyticdigestion (right-hand tube). KOH-based crème application forstraightening, a type of cosmetic treatment, causes hair to notdissolve, although the hair has been shown to be porous. This type ofhair is identified when, at 2 hours, the control hair is well dissolvedand the test hair is not dissolved (FIG. 3).

Example 6 Hair Integrity Test of Proteolytic Digests of Hair Samples:Cellophane Treatment

FIG. 4A is a photograph demonstrating a hair sample subjected tocellophane treatment prior to treatment using a proteolytic digestion(left-hand tube) and a control sample subjected treatment using aproteolytic digestion (right-hand tube). As shown in FIG. 4A, when ahair sample treated with cellophane containing a dye is placed in theproteolytic solution, within 5 minutes dye is visible in solution butthe hair may show the dissolution rate of control hair (FIG. 4A). Thiseffect is different from permanent dye treatments where the hair hasbeen damaged by the bleach/dye combination used for the treatment andtherefore dissolves and the sample will dissolve rapidly while releasingdye (FIG. 4B).

Example 7 Non-Proteolytic A₃₈₀ for Integrity Testing

Approximately 6 mg of hair was added to a plastic tube, followed by theaddition of DTT digest (1.2 mL) and shaking at 37o C for two hours.After this time period, 100 uL of the supernatant was removed from thetube and added to a 96 well microplate. 1 M HCl (10 uL) was then addedto the microplate wells, followed by measuring absorbance at awavelength of 380 nm (A₃₈₀) of the resulting solution. Results areprovided in Table 14 below and FIG. 6. Invalid samples (i.e., damageshair samples) were identified by having A380>0.35.

TABLE 14 Integrity A₃₈₀ 2 1.172 2 1.059 2 1.086 5 0.251 5 0.342 6 0.1666 0.171 8 0.184 8 0.093 8 0.175

Example 8 Proteolytic A₃₄₀ for Integrity Testing

Approximately 8 mg of hair was added to a plastic tube, followed by theaddition of proteolytic digest (1.2 mL) and shaking at 37o C. After 90minutes, 200 uL of solution was removed from the tube and added to amicroplate well. 1 M HCl (10 uL) was then added to the microplate well,followed by measuring the A340 of the resulting solution. Wells with thehigh absorbance corresponded to samples with low integrity values.Results are provided in FIG. 7.

Example 9 Use of Proteolytic Method to Compare Samples Submitted forFollow-Up Testing 9A. Loss of Analyte Due to Excessive Hair Damage:

A first hair sample collected from a subject contained 11.9 ng/10 mghair MDMA (ecstasy) and 0.7 ng/10 mg hair of MDA, the metabolite ofMDMA. Fifteen days later, a second hair sample was collected from saidsubject and sent for retesting. The second hair sample contained only0.3 ng MDMA/10 mg hair MDMA. Upon performance of the proteolyticintegrity test on both samples, it was found that the first hair samplesample did not dissolve until 2 hours, indicating it was not damaged.The second hair sample dissolved completely in 30 minutes, indicating itwas extremely damaged and porous. The second sample had been renderedinvalid for testing, either deliberately or without knowledge of thisoutcome by the subject.

9B. Excess Treatments:

A first hair sample collected from a subject contained 73 ng/10 mg haircocaine. Thirteen days later, a second hair sample was collected fromsaid subject and sent for retesting. The second hair sample contained0.3 ng cocaine/10 mg hair. Upon performance of the proteolytic integritytest on both samples, it was found that the first hair sample sampledissolved in 75 minutes, indicating it was somewhat damaged and porous,but not an invalid sample for testing. The second hair sample did notdissolve at all in 2 hours, indicating it had been relaxed with apotassium hydroxide cream treatment. Strong base treatment of hairrenders the hair impervious to the proteolytic digestion, although drugsare still eluted from the sample. Assuming the two hair samples camefrom the same subject as represented, performing a strong base treatmenton an already porous hair would cause it to be extremely damaged andporous such that the drug was eluted during the treatment and duringsubsequent regular shampooing. Results of second hair sample may bereported as invalid for testing.

9C. Loss of Analyte Due to Excessive Hair Damage).

A first hair sample collected from a subject contained 26.2 ng/10 mghair cocaine. Seven days later, a second hair sample was collected fromthe individual and sent for retesting, as a follow-up retest. The secondhair sample contained less than 5 ng cocaine/10 mg hair. Uponperformance of the proteolytic integrity test on both samples, it wasfound that the original sample dissolved in 75 minutes, indicating itwas somewhat damaged and porous, but not an invalid sample for testing.The second sample dissolved in 30 minutes, indicating it was a damagedand porous hair sample, causing the drug to be lost from the sample. Thesecond hair was reported as invalid for testing.

9D.

A hair sample contained 5.3 ng morphine/10 mg hair and 5.9 ng 6-MAM/10mg hair, indicating heroin ingestion. The sample also contained 36.2 pgcarboxy-THC (C-THC, marijuana metabolite)/10 mg hair. A sample collected9 days later contained 1.1 ng morphine/10 mg hair and 0.6 ng 6-MAM/10 mghair, negative for heroin use as the results are below the cutoff of 2ng/10 mg hair. The 2nd sample contained 14.8 ng C-THC. The first sampledissolved in 90 minutes (Integrity value=6) and the 2nd sample dissolvedin 45 minutes (integrity value=3). Although the heroin content becamenegative (lost 79% of the original content due to porosity of the hair),the marijuana content was still well above the positive level of 1 pg/10mg hair. Some analytes are more readily lost from hair than others. Ithas been the experience of the authors that cocaine and amphetamines aremost susceptible to loss from porosity, with opiates somewhat moreresistant, and marijuana most resistant to loss.

REFERENCES

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Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1.-22. (canceled)
 23. A method for determining the presence or absenceof a drug of abuse or metabolite thereof in a hair sample of a subjectcomprising: providing a hair sample; contacting the hair sample with anaqueous solution comprising a reducing agent and a protease capable ofdigesting keratin to result in a test sample, wherein the contactingstep occurs for a period of about 1 hour to about 2 hours; determiningthe amount of dissolved hair protein in the test sample using a proteinassay; identifying the hair in the test sample as not suitable for druganalyte testing when the hair in the test sample has dissolved in thetest sample more rapidly than the hair in a similarly contacted controlsample; and if the hair in the test sample is not identified as notsuitable for drug analyte testing, then determine if the drug of abuseor metabolite thereof is present or absent in the hair sample.
 24. Themethod of claim 23, wherein the protease is selected from the groupconsisting of papain, chymopapain, and proteinase K.
 25. The method ofclaim 23, further comprising assessing the condition of the hair in thetest sample by visual inspection at predetermined time intervals. 26.The method of claim 23, wherein the reducing agent is selected from thegroup consisting of 2,3 dihydroxybutane-1,4-dithiol (“DTT”), 2,3dihydroxybutane-1,4-dithiol (“DTE”), thioglycolate, cysteine, sulfites,bisulfites, sulfides, bisulfides, tris(2-carboxyethyl)phosphine(“TCEP”), and mixtures thereof.
 27. The method of claim 23, wherein theaqueous solution comprises 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9% or 1.0% of the reducing agent.
 28. The method of claim 23,wherein the pH at which the contacting step is performed is betweenabout 7.0 and 10.5, between about 8.8 and 10.5, between about 8.8 and9.7, between about 8.8 and 9.5, or between about 9.4 and 9.7
 29. Themethod of claim 23, wherein the temperature at which the contacting stepor the determining step is performed is between about 20° C. and about40° C.
 30. (canceled)
 31. The method of claim 23, wherein determiningthe amount of dissolved hair protein in the test sample occurs at about15 minute intervals.
 32. The method of claim 25, wherein thepredetermined time intervals are about 15 minute intervals.
 33. Themethod of claim 23, wherein the step of determining the amount ofdissolved hair protein in the test sample is completed using a portionof the hair sample; and determining the presence or absence of a drug ofabuse or metabolite thereof is completed using: (a) the same portion ofthe hair sample; or (b) another portion of the hair sample.
 34. Themethod of claim 23, further comprising identifying the hair sample asbeing previously treated with potassium hydroxide if the hair samplefails to dissolve after a time period of about 120 minutes.
 35. Themethod of claim 23, further comprising identifying the hair sample asbeing previously treated with a cellophane hair treatment if a dye isvisible in the test solution within about 5 minutes of contacting thehair sample with an aqueous solution comprising a reducing agent and aprotease.
 36. The method of claim 23, wherein the hair sample is awashed hair sample.
 37. The method of claim 23, wherein the amount ofdissolved hair protein the test sample is determined using a proteinassay selected from the group consisting of the Lowry Assay or theBradford Assay.
 38. The method of claim 23, further comprisingindicating the hair sample as unsuitable for determining if an analyteis present in the test solution if the hair sample has been identifiedas damaged.
 39. The method of claim 23, wherein determining the presenceor absence of a drug of abuse or a metabolite thereof comprises anenzyme immunoassay specific for the analyte, a mass spectrometrytechnique, or a chromatographic technique.
 40. The method of claim 23,wherein the drug of abuse or metabolite thereof is selected from thegroup consisting of a prescription medicine or metabolite thereof, apain medication or metabolite thereof, a nutrient, and an endogenousanalyte, or a salt form of any of the foregoing.
 41. The method of claim40, wherein the drug of abuse or metabolite thereof is selected from thegroup consisting of cocaine, benzoylecgonine, cocaethylene, norcocaine,PCP, amphetamine, methamphetamine, cannabinoid, THC, carboxy-THC,heroin, codeine, morphine, 6-monoacetylmorphine (MAM), oxycodone,3,4-methylenedioxyamphetamine (MDA) and3,4-methylenedioxymethamphetamine (MDMA).
 42. The method of claim 40,wherein the prescription medicine or metabolite thereof, or painmedication or metabolite thereof is an opioid, cannabinoid, NSAID,steroid, amphetamine, benzodiazepine, barbiturate, tricyclic, orephedrine, or metabolite thereof.
 43. The method of claim 23, furthercomprising determining the amount of the analyte in the test solution.44. The method of claim 23, wherein determining the amount of dissolvedhair protein in the test sample occurs at 0, 15, 30, 45, 60, 75, 90 and120 minutes after contacting the hair sample with an aqueous solutioncomprising a reducing agent and a protease capable of digesting keratin.45. The method of claim 23, further comprising identifying the hairsample as suitable for further processing if the rate of dissolution inthe test solution is commensurate with the rate of dissolution in thecontrol sample.
 46. The method of claim 23, further comprisingdetermining the rate of dissolution of the hair in the test sample usinga protein assay; and identifying the hair in the test sample as notsuitable for drug analyte testing when the hair in the test sample hasdissolved in the test sample more rapidly than the hair in a similarlycontacted control sample.
 47. A method for determining the presence orabsence of a drug of abuse or metabolite thereof in a hair sample of asubject comprising: providing a hair sample; contacting the hair samplewith an aqueous solution comprising a reducing agent and a proteasecapable of digesting keratin to result in a test sample, wherein thecontacting step occurs for a period of about 1 hour to about 2 hours;determining the rate of dissolution of the hair in the test sample usinga protein assay; and identifying the hair in the test sample as notsuitable for drug analyte testing when the hair in the test sample hasdissolved in the test sample more rapidly than the hair in a similarlycontacted control sample; and if the hair in the test sample is notidentified as not suitable for drug analyte testing, then determine ifthe drug of abuse or metabolite thereof is present or absent in the hairsample.